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Identification and Validation of FMRP Target RNAs

FMRP 目标 RNA 的鉴定和验证

基本信息

批准号：
7932656
负责人：
JENNIFER C DARNELL
金额：
$ 17.17万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7932656
关键词：
Address Affect Antibodies Behavioral Assay Binding Binding Sites Biochemical Bioinformatics Biological Assay Biological Models Boxing Brain Circadian Rhythms Complement Complex Conditioned Reflex Data Dependence Disease Dose FMR1 FMRP FXR2 gene Failure Foundations Fragile X Mental Retardation Protein Fragile X Syndrome G-Quartets Gel Goals Histology Human Immunofluorescence Immunologic In Situ Hybridization In Vitro KH Domain Knock-in Mouse Knockout Mice Learning Ligands Long-Term Depression Maps Measures Mental Retardation Messenger RNA Metabolism Methods Microarray Analysis Missense Mutation Mus Mutagenesis Mutation Nature Neurologic Neurons Patients Phenotype Polyribosomes Protein Biosynthesis Proteins RNA RNA Binding RNA Recognition Motif RNA Sequences RNA analysis RNA-Binding Proteins Regulation Reporter Reverse Transcriptase Polymerase Chain Reaction Role Structure Sucrose Testing Translational Regulation Translations Validation Western Blotting crosslink improved in vivo in vivo Model mouse model mutant nucleotide analog research study tissue/cell culture validation studies

项目摘要

DESCRIPTION (provided by applicant): This is a proposal to test the hypothesis that Fragile-X mental retardation results from a failure of FMRP to bind specific RNAs, and to identify those RNAs. The foundation of this proposal is our finding of sequence and structure-specific targets for the FMRP RGG box and KH2 domain, the latter of which is associated with severe disease in a patient with a missense mutation (I304N) in this RNA-binding domain. We have related the KH2 RNA target, termed a kissing complex RNA, to disease by demonstrating that it competes FMRP off of brain polyribosomes, suggesting that this RNA motif is used by FMRP to effect translational regulation of specific mRNAs. Three aspects of FMRP sequence-specific RNA binding will be definitively addressed here. First, FMRP RNA targets will be identified using several methods: bioinformatic screens complemented by structural studies, microarray analysis of coimmunoprecipitating RNAs, microarray analysis of RNAs whose distribution on polyribosomes is shifted in the absence of FMRP, and cross-linking IP (CLIP) studies. Second, to help validate these new RNA targets in vivo, we will generate three new mouse models of Fragile-X mental retardation. Third, these new model systems will be used in conjunction with the existing FMR1-null mouse to validate identified RNA targets of FMRP in mouse brain. Taken together, these studies will address the hypothesis that mental retardation associated with the I304N mutation, and likely the Fragile-X syndrome more generally, may relate to a crucial role for RNAs harboring the kissing complex motif as targets for FMRP translational regulation.

描述（由申请人提供）：这是一个测试脆性X智力低下是由FMRP未能结合特定RNA引起的假设的建议，并鉴定这些RNA。这一建议的基础是我们发现了FMRP RGG盒和KH 2结构域的序列和结构特异性靶点，后者与该RNA结合结构域中存在错义突变（I304 N）的患者的严重疾病相关。我们已经将KH 2 RNA靶点（称为接吻复合物RNA）与疾病联系起来，证明它与脑多聚核糖体中的FMRP竞争，表明FMRP使用该RNA基序来影响特定mRNA的翻译调控。FMRP序列特异性RNA结合的三个方面将在这里明确解决。首先，FMRP RNA靶标将使用几种方法来鉴定：生物信息学筛选，辅以结构研究，免疫共沉淀RNA的微阵列分析，在不存在FMRP的情况下其在多聚核糖体上的分布发生变化的RNA的微阵列分析，以及交联IP（CLIP）研究。其次，为了帮助在体内验证这些新的RNA靶点，我们将产生三种新的脆性X智力低下小鼠模型。第三，这些新的模型系统将与现有的FMR 1-null小鼠结合使用，以验证小鼠大脑中FMRP的已鉴定RNA靶标。总之，这些研究将解决与I304 N突变相关的精神发育迟滞，以及更普遍的脆性X综合征可能与携带接吻复合体基序的RNA作为FMRP翻译调控靶点的关键作用有关的假设。