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: 8506192
• 负责人: JENNIFER C DARNELL
• 金额: $ 35.17万
• 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/8506192
• 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; 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综合征（FXS）是由三重重复扩增引起的，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会导致FXS特征性的智力残疾、癫痫发作和自闭症特征。FMRP被广泛接受为调节特定mRNA的翻译，并且神经元中的许多形式的突触可塑性依赖于其功能。脆性X患者在FMRP的RNA结合结构域内发生的错义突变足以引起疾病并废除FMRP的多核糖体缔合，这表明识别FMRP的mRNA靶点以及FMRP如何调节其翻译是理解该疾病典型的认知和行为变化的分子基础的关键。设计用于捕获RNA结合蛋白与其RNA靶标的体内相互作用的新技术，UV交联-免疫沉淀结合高通量测序（HITS-CLIP），用于鉴定全脑多聚核糖体中FMRP相互作用的mRNA，所述全脑多聚核糖体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。发现FMRP调节编码对突触功能重要的突触前和突触后蛋白的mRNA，这表明在FMRP不存在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靶点的清单是一个资源，用于集中研究，通过抑制可能在FMRP缺乏时过度表达的蛋白质的活性来改善FXS的症状。为了鉴定与表型缺陷最相关的FMRP靶标的子集，该提议旨在通过工程化新小鼠（cTAG）来开发和应用测量FMRP与RNA的细胞特异性相互作用的创新方法，所述新小鼠（cTAG）将通过与诱导型Cre系育种从具有时间控制的特定细胞中的内源基因座有条件地标记FMRP，使得所述标签可用于HITS-CLIP。 FXS是由于FMRP在其两个家族成员FXR 1 P和FXR 2 P正常表达的情况下丢失而产生的，这两个家族成员与FMRP共享一些功能冗余。这导致了一个根本性的问题，即FXS是否是由于FMRP特异性功能的丧失而不是其旁系同源物所共有的，或者FXS是否是由于功能冗余蛋白家族的剂量减少而引起的。该提案将测试三种旁系同源物在RNA相互作用方面的体内功能冗余，包括全局和特定神经元中与FXR 1/2 P cTAG小鼠的相互作用，并测试在FMRP不存在的情况下增加FXR 1/2 P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。FMRP的特异性、表型相关mRNA靶点的鉴定不被其旁系同源物共享，这将集中注意力于抑制该亚群，而冗余的证据将刺激尝试治疗上调FXS中FXR 1/2 P蛋白的水平。