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

描述(申请人提供)：脆性X综合征(FXS)是由一个三联体重复扩增引起的，其异常甲基化导致脆性X智力低下蛋白FMRP的表达沉默。这种神经元RNA结合蛋白功能的丧失会导致FXS特有的智能障碍、癫痫发作和自闭症特征。FMRP被广泛接受来调节特定mRNAs的翻译，神经元中许多形式的突触可塑性依赖于它的功能。在脆性X患者中，FMRP的RNA结合域中发生的错义突变足以导致该病，并取消FMRP的多聚体关联，这表明识别FMRP的mRNA靶点以及FMRP如何调节它们的翻译是理解该病典型认知和行为变化的分子基础的关键。一种旨在捕捉RNA结合蛋白与其RNA靶标之间的体内相互作用的新技术，即紫外光交联免疫沉淀结合高通量测序(HITS-CLIP)，被用于在整个脑多聚体中识别与FMRP相互作用的mRNAs，其中包括处于不同发育或活动状态的多种神经元亚型的混合物。FMRP被发现调节编码突触前和突触后蛋白的mRNAs，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原因。这份842个FMRP靶点的清单是一个资源，用于集中研究通过抑制在没有FMRP的情况下可能过度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靶子集，本建议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，方法是设计一种新的小鼠(CTAG)，它将通过与可诱导的Cre系繁殖来有条件地标记特定细胞中具有时间控制的内源基因的FMRP，从而使该标记可用于HITS-CLIP。FXS是在其两个家族成员FXR1P和FXR2P正常表达的情况下由于FMRP的缺失而产生的，这两个家族成员与FMRP有一些功能冗余。这导致了一个根本的问题，即FXS是由于FMRP特有的而不是其同源基因所共享的功能的丧失，还是由于一系列功能冗余蛋白质的剂量减少所致。这项建议将在体内测试这三个近似体与FXR1/2P CTAG小鼠的RNA相互作用的功能冗余，并测试在缺乏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突触功能障碍导致的人类疾病，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这一亚群上，而冗余的证据将刺激尝试在治疗上上调FXR1/2P蛋白在FXS中的水平。

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.