RNA Targets for Fragile X Mental Retardation Protein

脆性 X 智力迟钝蛋白的 RNA 靶标

• 批准号: 9235006
• 负责人: Alexander Serganov
• 金额: $ 21.19万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-26 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9235006
• 关键词: Affect; Affinity; Anabolism; Animal Model; Autistic Disorder; Binding; Binding Proteins; Binding Sites; Biochemical; Bioinformatics; Biological Assay; Brain; Categories; Cell physiology; Characteristics; Conflict (Psychology); Data; Data Set; Defect; Development; Disease; Elements; FMR1; Fragile X Syndrome; Genetic; Genetic Translation; Goals; Human; Human Activities; Inherited; Intellectual functioning disability; KH Domain; Knowledge; Learning; Left; Mediating; Medical; Memory; Mental Retardation; Messenger RNA; Methods; Mission; Molecular; Mutation; Nervous System Physiology; Neurologic; Neurons; Pathogenesis; Play; Process; Protein Biosynthesis; Protein Engineering; Proteins; Public Health; Publishing; RNA; RNA Binding; RNA Recognition Motif; RNA Sequences; RNA-Binding Proteins; RNA-Protein Interaction; Reporting; Research Personnel; Ribosomes; Roentgen Rays; Role; Specificity; Structure; Synapses; Synaptic plasticity; Syndrome; Tertiary Protein Structure; Testing; Therapeutic; Translational Repression; Translations; United States National Institutes of Health; Untranslated RNA; Variant; X-Ray Crystallography; base; combinatorial; design; functional loss; high reward; high risk; human disease; impression; insight; long term memory; mutant; neurological pathology; next generation sequencing; novel; novel strategies; novel therapeutic intervention; protein function; research study

Summary mRNA-binding proteins play a pivotal role in the development and function of the nervous system and defects in the function of these proteins underlie a broad spectrum of neurological pathologies. Fragile X Mental Retardation Protein (FMRP) is a paradigm of disease-associated RNA-binding proteins because of its essential contribution to the development and activity of the brain and its central role in several human disorders that affect hundreds of thousands people. The loss of FMRP due to transcriptional silencing or protein mutations leads to Fragile X syndrome (FXS), a common familial cause of inherited intellectual disability and autism that currently lacks an efficient medical treatment. On the molecular level, the absence of functional FMRP results in exaggerated protein biosynthesis that is normally held in check by FMRP-mediated translational repression of selected mRNAs. Previous studies have reported mostly conflicting datasets of FMRP targets, and despite its vital importance, the mechanism of mRNA selection by FMRP remains unclear. This lack of definitive knowledge on the principles of FMRP-RNA recognition limits both understanding of FXS and the development of rational therapeutic approaches for its treatment. Our preliminary structural data suggest that FMRP can bind RNA in sequence-specific manner and that RNA binding of FMRP is not truly promiscuous. The objective of this proposal is to determine specific RNA targets for human FMRP and understand the molecular principles of FMRP-RNA recognition. The hypothesis is that RNA-binding domains of FMRP recognize RNA sequence- specifically and that combinations of these RNA motifs determine binding to natural RNAs. To test this hypothesis, FMRP binding sites will be identified using a novel biochemical approach and structural studies. Specific Aim 1 is devoted to identification of short RNA sequences that bind specifically to isolated KH domains of FMRP by using a novel “bottom-up” approach that combines RNA capture experiments with Next Generation Sequencing. Specific Aim 2 will characterize the molecular features of FMRP that are essential for specific RNA binding by using biochemical methods and X-ray crystallography. Specific Aim 3 will aim to develop mutant FMRP proteins with altered RNA specificity to study various FMRP functions. Together, these results will define RNA sequence elements required for interactions with FMRP, help to identify natural RNA targets of FMRP, and design mutant FMRP proteins to interrogate various FMRP functions in the animal models of FXS. The proposal is highly relevant to public health and the NIH mission since it will provide insights on the RNA recognition and the mechanism of FMRP-mediated translational inhibition, the activities associated with development of FXS, autism and other disorders. Understanding how FMRP functions will advance searches for novel therapeutic interventions against FXS and related diseases.

概括 mRNA结合蛋白在神经系统的发育和功能以及缺陷中发挥着关键作用 这些蛋白质的功能是广泛的神经病理学的基础。脆弱X精神 延迟蛋白 (FMRP) 是疾病相关 RNA 结合蛋白的一个范例，因为它具有重要的作用 对大脑发育和活动的贡献及其在多种人类疾病中的核心作用 影响数十万人。由于转录沉默或蛋白质突变导致 FMRP 丢失 导致脆性 X 综合征 (FXS)，这是遗传性智力障碍和自闭症的常见家族原因， 目前缺乏有效的医疗手段。在分子水平上，缺乏功能性 FMRP 结果 过度的蛋白质生物合成通常受到 FMRP 介导的翻译抑制的控制 选定的 mRNA。先前的研究报告了 FMRP 目标的数据集大多相互矛盾，尽管 尽管 FMRP 选择 mRNA 的重要性仍不清楚，但其机制仍不清楚。这种缺乏确定性的 对 FMRP-RNA 识别原理的了解限制了对 FXS 的理解和开发 其治疗的合理治疗方法。我们的初步结构数据表明 FMRP 可以 FMRP 以序列特异性方式结合 RNA，并且 FMRP 的 RNA 结合并不是真正混杂的。目标 该提案的目的是确定人类 FMRP 的特定 RNA 靶标并了解分子原理 FMRP-RNA 识别。假设 FMRP 的 RNA 结合域识别 RNA 序列 - 具体而言，这些 RNA 基序的组合决定了与天然 RNA 的结合。为了测试这个 假设，FMRP 结合位点将使用新的生化方法和结构研究来鉴定。 具体目标 1 致力于鉴定与分离的 KH 结构域特异性结合的短 RNA 序列 FMRP 通过使用一种新颖的“自下而上”方法将 RNA 捕获实验与 Next 相结合 世代测序。具体目标 2 将描述 FMRP 的分子特征，这些特征对于 使用生化方法和 X 射线晶体学进行特异性 RNA 结合。具体目标 3 将旨在 开发具有改变的 RNA 特异性的突变 FMRP 蛋白来研究各种 FMRP 功能。在一起，这些 结果将定义与 FMRP 相互作用所需的 RNA 序列元件，有助于识别天然 RNA FMRP 的靶标，并设计突变 FMRP 蛋白来探究动物中的各种 FMRP 功能 FXS 型号。该提案与公共卫生和 NIH 使命高度相关，因为它将提供 对 RNA 识别和 FMRP 介导的翻译抑制机制的见解、活性 与 FXS、自闭症和其他疾病的发展有关。了解 FMRP 的运作方式 提前寻找针对 FXS 及相关疾病的新型治疗干预措施。