RNA Targets for Fragile X Mental Retardation Protein

脆性 X 智力迟钝蛋白的 RNA 靶标

• 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
• 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

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.

总结 mRNA结合蛋白在神经系统的发育和功能缺陷中起着关键作用 这些蛋白质的功能是广泛的神经病理学的基础。脆性X智力 阻滞蛋白（FMRP）是疾病相关RNA结合蛋白的一个范例，因为它具有重要的生物学功能。 对大脑发育和活动的贡献及其在几种人类疾病中的核心作用， 影响了成千上万的人由于转录沉默或蛋白质突变导致的FMRP缺失 导致脆性X综合征（FXS），这是遗传性智力残疾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，功能性FMRP的缺乏导致 在通常由FMRP介导的翻译抑制控制的过度蛋白质生物合成中， 选择的mRNA。以前的研究报告了FMRP目标的大多数相互冲突的数据集，尽管 尽管其至关重要，但FMRP选择mRNA的机制仍不清楚。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FXS的发展。 合理的治疗方法。我们的初步结构数据表明，FMRP可以 以序列特异性方式结合RNA，并且FMRP的RNA结合不是真正混杂的。客观 该方案的一个重要目的是确定人FMRP的特异性RNA靶点， FMRP-RNA识别。假设是FMRP的RNA结合结构域识别RNA序列- 这些RNA基序组合决定了与天然RNA的结合。为了验证这一 假设，FMRP结合位点将使用新的生物化学方法和结构研究来鉴定。 特异性目标1致力于鉴定特异性结合分离的KH结构域的短RNA序列 通过使用一种新的“自下而上”的方法，将RNA捕获实验与Next 世代排序。具体目标2将描述FMRP的分子特征，这些特征对于 通过使用生物化学方法和X-射线晶体学的特异性RNA结合。具体目标3旨在 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。所有这些 结果将定义与FMRP相互作用所需的RNA序列元件，有助于鉴定天然RNA FMRP的靶点，并设计突变的FMRP蛋白质以询问动物中的各种FMRP功能 FXS模型该提案与公共卫生和NIH的使命高度相关，因为它将提供 对RNA识别和FMRP介导的翻译抑制机制的见解， 与FXS、自闭症和其他疾病的发展有关。了解FMRP的功能将 针对FXS和相关疾病的新型治疗干预的先进搜索。