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

脆性 X 智力迟钝蛋白家族的细胞特异性 RNA 靶标

• 批准号: 8625795
• 负责人: JENNIFER C DARNELL
• 金额: $ 34.19万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-15 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8625795
• 关键词: Address; Adult; Angelman Syndrome; Attention; Autistic Disorder; Behavioral; Binding; Biological Assay; Brain; Breeding; Candidate Disease Gene; Cell Differentiation process; Cell Lineage; Cell Proliferation; Cells; Characteristics; Cognition Disorders; Cognitive; Data; Defect; Development; Disease; Disease model; Engineering; Equilibrium; Excitatory Synapse; Exhibits; FMRP; FXR1 gene; Family; Family member; Fragile X Mental Retardation Protein; Fragile X Syndrome; Functional disorder; Funding; Future; Gene Family; Genes; Genetic; Goals; High-Throughput Nucleotide Sequencing; Hippocampus (Brain); Human; Immunoprecipitation; In Vitro; Intellectual functioning disability; Intention; Knock-in Mouse; Lead; Ligands; Measures; Mental Retardation; Messenger RNA; Methylation; Missense Mutation; Molecular; Molecular Probes; Morphology; Mus; Neurons; Patients; Phase; Phenotype; Physiological; Polyribosomes; Population; Process; Property; Protein Family; Proteins; Proteome; Pyramidal Cells; RNA; RNA Binding; RNA Recognition Motif; RNA-Binding Proteins; Research; Resources; Ribosomes; Role; Seizures; Symptoms; Synapses; Synaptic plasticity; System; Techniques; Technology; Test Result; Testing; Time; Translational Regulation; Translations; Trinucleotide Repeats; Up-Regulation; Validation; Vertebral column; autism spectrum disorder; base; cell type; crosslink; dentate gyrus; design; dosage; human disease; improved; in vivo; innovation; insight; loss of function; mouse model; nerve stem cell; new technology; overexpression; paralogous gene; postsynaptic; presynaptic; protein complex; protein degradation; protein function; public health relevance; research study; stem; stoichiometry; synaptic function

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.

描述（由申请人提供）：脆性 X 染色体综合征 (FXS) 是由三联体重复扩增引起的，其异常甲基化使脆性 X 染色体智力迟钝蛋白 FMRP 的表达沉默。这种神经元 RNA 结合蛋白的功能丧失会导致 FXS 特有的智力障碍、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都依赖于其功能。脆性 X 患者中 FMRP RNA 结合域内发生的错义突变足以引起疾病并消除 FMRP 的多核糖体关联，这表明识别 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 靶标的体内相互作用的新技术，即紫外交联免疫沉淀结合高通量测序 (HITS-CLIP)，用于识别全脑多聚体中的 FMRP 相互作用 mRNA，包括处于不同发育或活动状态的许多神经元亚型的混合物。 FMRP被发现可以调节编码对突触功能重要的突触前和突触后蛋白质的mRNA，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。这份包含 842 个 FMRP 靶点的列表是集中研究的资源，通过抑制在缺乏 FMRP 的情况下可能过度表达的蛋白质的活性来改善 FXS 症状。为了确定与表型缺陷最相关的 FMRP 靶标子集，本提案旨在开发和应用一种创新方法，通过工程设计一种新小鼠 (cTAG) 来测量 FMRP 与 RNA 的细胞特异性相互作用，该小鼠将通过与诱导型 Cre 系育种，有条件地标记来自特定细胞内源基因座的 FMRP，并进行时间控制，以便该标签可用于 HITS-CLIP。 FXS 的产生是由于 FMRP 在其两个家族成员 FXR1P 和 FXR2P 正常表达的情况下丢失，这两个家族成员与 FMRP 共享一些功能冗余。这就引出了一个基本问题：FXS 是否是由于 FMRP 特有的功能丧失而引起的，并且其旁系同源物不具有这种功能，或者 FXS 是否是由于功能冗余蛋白家族的剂量减少所致。该提案将在 FXR1/2P cTAG 小鼠的整体和特定神经元中测试三种旁系同源物在 RNA 相互作用方面的体内功能冗余，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。成功实现所提出的目标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。鉴定与其旁系同源物不共享的 FMRP 特定的、与表型相关的 mRNA 靶标，将把注意力集中在对该子集的抑制上，而冗余的证据将刺激尝试在治疗上上调 FXS 中 FXR1/2P 蛋白的水平。