RNA Targets for Fragile X Mental Retardation Protein

脆性 X 智力迟钝蛋白的 RNA 靶标

基本信息

批准号：
9357716
负责人：
Alexander Serganov
金额：
$ 25.43万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-26 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9357716
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)Crystallization Data Data Set Defect Development Disease Elements FMR1 Fragile X Syndrome Genetic Genetic Transcription 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 Ribosomal Proteins 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 experimental study 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

项目摘要

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多种神经病理学的基础。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由于转录沉默或蛋白质突变而导致 FMRP 丧失，影响了数十万人。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，目前缺乏有效的医学治疗方法，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先前的研究报告了 FMRP 目标的大部分相互矛盾的数据集，尽管 FMRP 选择 mRNA 的机制至关重要，但目前尚不清楚。对 FMRP-RNA 识别原理的了解限制了对 FXS 的理解和开发我们的初步结构数据表明 FMRP 可以治疗该病。 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。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 识别的假设是 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。假设，FMRP 结合位点将通过一种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 相结合来实现 FMRP 生成测序。特定目标 2 将表征 FMRP 所必需的分子特征。具体目标 3 将通过使用生化方法和 X 射线晶体学来实现特异性 RNA 结合。开发具有改变的 RNA 特异性的突变 FMRP 蛋白，以研究各种 FMRP 功能。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 的靶点，并设计突变 FMRP 蛋白来探究动物中的各种 FMRP 功能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、活性了解 FMRP 的功能与 FXS、自闭症和其他疾病的发展相关。寻找针对 FXS 和相关疾病的新型治疗干预措施取得了进展。