Identification and Validation of FMRP Target RNAs

FMRP 目标 RNA 的鉴定和验证

• 批准号: 7799774
• 负责人: JENNIFER C DARNELL
• 金额: $ 27.86万
• 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 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7799774
• 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

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）：本提案旨在检验 Fragile-X 精神发育迟滞是由于 FMRP 未能结合特定 RNA 造成的假设，并鉴定这些 RNA。该提案的基础是我们发现了 FMRP RGG 盒和 KH2 结构域的序列和结构特异性靶标，后者与该 RNA 结合结构域中存在错义突变 (I304N) 的患者的严重疾病相关。我们通过证明 KH2 RNA 靶标（称为接吻复合体 RNA）与大脑多核糖体竞争 FMRP，将其与疾病联系起来，表明 FMRP 使用该 RNA 基序来影响特定 mRNA 的翻译调节。这里将明确讨论 FMRP 序列特异性 RNA 结合的三个方面。首先，FMRP RNA靶标将使用多种方法进行鉴定：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、在没有FMRP的情况下对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 靶标，我们将生成三种新的 Fragile-X 智力障碍小鼠模型。第三，这些新模型系统将与现有的 FMR1-null 小鼠结合使用，以验证小鼠大脑中已识别的 FMRP RNA 靶标。总而言之，这些研究将提出这样一个假设：与 I304N 突变相关的精神发育迟滞，以及更普遍的脆性 X 综合征，可能与含有接吻复合体基序的 RNA 作为 FMRP 翻译调节的靶点的关键作用有关。