Cell-specific RNA Targets of the Fragile X Mental Retardation Protein Family

描述（由申请人提供）：脆性X综合征（FXS）是由三联体重复扩增引起的，其异常甲基化使脆性X智力迟钝蛋白（FMRP）的表达沉默。这种神经元RNA结合蛋白的功能丧失导致FXS的智力残疾、癫痫发作和自闭症特征。FMRP被广泛认为可以调节特定mrna的翻译，神经元中许多形式的突触可塑性依赖于它的功能。脆性X患者在FMRP的RNA结合区域内发生的错义突变足以导致该疾病并消除FMRP的多体关联，这表明确定FMRP的mRNA靶点以及FMRP如何调节其翻译是了解该疾病典型认知和行为变化的分子基础的关键。一种旨在捕获RNA结合蛋白与其RNA靶点的体内相互作用的新技术，紫外交联免疫沉淀结合高通量测序（HITS-CLIP），用于鉴定包括不同发育或活性状态的许多神经元亚型混合物在内的总脑多体中FMRP相互作用的mrna。研究发现，FMRP可以调节编码突触前和突触后蛋白的mrna，这些蛋白对突触功能至关重要，这表明在缺乏FMRP的情况下，它们的错误表达可能是突触可塑性缺陷的基础。这份842个FMRP靶点列表是重点研究的资源，可以通过抑制在缺乏FMRP时可能过表达的蛋白质的活性来改善FXS的症状。为了确定与表型缺陷最相关的FMRP靶点子集，本提案旨在开发和应用一种创新的方法来测量FMRP与RNA的细胞特异性相互作用，方法是通过设计一种新的小鼠（cTAG），该小鼠将有条件地标记FMRP，标记来自特定细胞中的内源性位点，并通过诱导Cre系进行时间控制，以便该标记可用于HITS-CLIP。FXS是由于FMRP的两个家族成员FXR1P和FXR2P在正常表达的情况下失去了FMRP而产生的，FXR1P和FXR2P与FMRP有一定的功能冗余。这就导致了一个基本问题：FXS是由于FMRP特有的功能丧失而引起的，而FMRP的类似物却没有这种功能，还是FXS是由于功能冗余蛋白家族的剂量减少而引起的。本研究将在体内测试三种类似物在RNA相互作用方面的功能冗余性，包括全局和特定神经元与FXR1/2P cTAG小鼠的相互作用，并测试在没有FMRP的情况下增加FXR1/2P的表达是否可以挽救表型。这些目标的成功实现对于理解突触功能的分子基础以及由其功能障碍引起的人类疾病，包括脆性X综合征和自闭症具有重要意义。鉴定FMRP的特异性、表型相关的mRNA靶点，将把注意力集中在抑制该亚群上，而冗余的证据将刺激尝试上调FXS中FXR1/2P蛋白的水平。

DESCRIPTION (provided by applicant): Fragile X Syndrome (FXS) is caused by a triplet repeat expansion whose abnormal methylation silences expression of the Fragile X Mental Retardation protein, FMRP. Loss of function of this neuronal RNA- binding protein results in the intellectual disability, seizures and autistic features characteristic of FXS. FMRP is widely accepted to regulate translation of specific mRNAs and many forms of synaptic plasticity in neurons are dependent on its function. A missense mutation occurring in a Fragile X patient within an RNA binding domain of FMRP is sufficient to cause the disease and abolishes FMRP's polysome association, suggesting that identifying FMRP's mRNA targets and how FMRP regulates their translation is key to understanding the molecular basis for the cognitive and behavioral changes typical of the disease. A new technique designed to capture in vivo interactions of RNA binding proteins with their RNA targets, UV crosslinking- immunoprecipitation combined with high throughput sequencing (HITS-CLIP), was used to identify FMRP- interacting mRNAs in total brain polysomes including a mixture of many neuronal subtypes in different states of development or activity. FMRP was found to regulate mRNAs encoding both pre- and postsynaptic proteins important for synaptic function, suggesting that their mis-expression in the absence of FMRP might underlie defects in synaptic plasticity. This list of 842 FMRP targets is a resource for focusing research to ameliorate symptoms of FXS by inhibiting the activity of proteins that may be overexpressed in the absence of FMRP. To identify the subset of targets of FMRP that are most relevant for phenotypic defects this proposal aims to develop and apply an innovative approach to measuring cell-specific interactions of FMRP with RNA by engineering a new mouse (cTAG) which will conditionally tag FMRP from the endogenous locus in specific cells with temporal control by breeding with inducible Cre lines so that the tag can be used for HITS-CLIP. FXS arises due to loss of FMRP in the context of normal expression of its two family members, FXR1P and FXR2P, which share some functional redundancy with FMRP. This has led to the fundamental question of whether FXS arises due to loss of a function specific to FMRP and not shared by its paralogs, or whether FXS results from decreased dosage of a family of functionally redundant proteins. This proposal will test in vivo functional redundancy of the three paralogs with regard to RNA interactions, both globally and in specific neurons with FXR1/2P cTAG mice, and test whether increasing expression of FXR1/2P in the absence of FMRP can rescue phenotype. Successful accomplishment of the proposed Aims has significance for understanding the molecular basis of synaptic function as well as the human diseases that result from its dysfunction, including Fragile X Syndrome and autism. Identification of specific, phenotype-relevant mRNA targets of FMRP not shared by its paralogs will focus attention on inhibition of this subset while evidence for redundancy will spur attempts to therapeutically upregulate the levels of the FXR1/2P proteins in FXS.