RNA-protein interactions in Fragile X syndrome

脆性 X 综合征中的 RNA-蛋白质相互作用

基本信息

批准号：
8686165
负责人：
Alexander Serganov
金额：
$ 16.78万
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：
2013
资助国家：
美国
起止时间：
2013-09-26 至 2015-09-25
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8686165
关键词：
Address Affinity Anabolism Autistic Disorder Base Sequence Binding Binding Proteins Binding Sites Biochemical Cell physiology Cleaved cell Code Cognitive Complement Complementary DNA Complex Computer Simulation Controlled Environment Data Defect Development Disease Elements Ensure Environment Epitopes Fragile X Syndrome Freezing Funding Future Gene Proteins Genetic Translation Goals Guanine Human Pathology Inherited Intellectual functioning disability KH Domain Laboratories Length Malignant Neoplasms Maps Mediating Medical Medical center Mental Retardation Messenger RNA Metabolic Diseases Methodology Mission Molecular Molecular Conformation Molecular Target Mus Mutation Myopathy Nerve Degeneration Neurons Patient Care Phase Pilot Projects Polyribosomes Premature Ovarian Failure Printing Protein Binding Protein Footprinting Proteins Public Health RNA RNA Binding RNA Recognition Motif RNA-Binding Proteins RNA-Protein Interaction Reagent Recovery Research Research Project Grants Sampling Science Site Specificity Structure Synapses Syndrome Techniques Temperature Testing Time Translational Repression Translations United States National Institutes of Health Ursidae Family Variant X-Ray Crystallography base experience foot genome-wide human disease improved in vitro testing insight knowledge base loss of function novel novel therapeutic intervention nuclease prevent programs protein complex protein function public health relevance repaired research study restoration synaptic function

Address Affinity Anabolism Autistic Disorder Base Sequence Binding Binding Proteins Binding Sites Biochemical Cell physiology Cleaved cell Code Cognitive Complement Complementary DNA Complex Computer Simulation Controlled Environment Data Defect Development Disease Elements Ensure Environment Epitopes Fragile X Syndrome Freezing Funding Future Gene Proteins Genetic Translation Goals Guanine Human Pathology Inherited Intellectual functioning disability KH Domain Laboratories Length Malignant Neoplasms Maps Mediating Medical Medical center Mental Retardation Messenger RNA Metabolic Diseases Methodology Mission Molecular Molecular Conformation Molecular Target Mus Mutation Myopathy Nerve Degeneration Neurons Patient Care Phase Pilot Projects Polyribosomes Premature Ovarian Failure Printing Protein Binding Protein Footprinting Proteins Public Health RNA RNA Binding RNA Recognition Motif RNA-Binding Proteins RNA-Protein Interaction Reagent Recovery Research Research Project Grants Sampling Science Site Specificity Structure Synapses Syndrome Techniques Temperature Testing Time Translational Repression Translations United States National Institutes of Health Ursidae Family Variant X-Ray Crystallography base experience foot genome-wide human disease improved in vitro testing insight knowledge base loss of function novel novel therapeutic intervention nuclease prevent programs protein complex protein function public health relevance repaired research study restoration synaptic function

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项目摘要

DESCRIPTION (provided by applicant): Defects in the function of mRNA-binding proteins underlie a broad spectrum of human pathologies. Fragile Mental Retardation Protein (FMRP) is one of these mRNA-binding proteins, involved in transport, storage and control of mRNA; changes in its biosynthesis cause several developmental and cognitive deficiencies including premature ovarian failure and fragile X syndrome (FXS). FXS is the most common inherited form of intellectual disability and a predominant monogenic cause of autism. The syndrome is associated with transcriptional silencing of the FMRP gene or a mutation in an RNA-binding domain of FMRP. On the molecular level, FXS is likely caused by dysregulation of FMRP-mediated translational control in neurons leading to altered synaptic function and other abnormalities. Despite identification of many FMRP-associated mRNAs by various approaches, the basis for mRNA recognition by FMRP is still not understood. The objective of this proposal is to restore the pilot research project focused on elucidating the principles of mRNA recognition by FMRP. The hypothesis is that FMRP recognizes mRNA targets that bear both sequence and structure-specific binding determinants. To test this hypothesis, it is proposed to identify FMRP binding sites using novel methodology and characterize FMRP-RNA interactions biochemically and biophysically. Specific Aim 1 is devoted to development of genome-wide RNA foot printing that implements conformation-specific nucleases with complementary specificities to identify FMRP-binding sites and probe their conformation. The mRNA structure will be tested in vitro by nuclease cleavage of total mouse mRNA in the absence and presence of FMRP. The cleaved mRNA fragments will be converted to cDNA and sequenced. FMRP binding sites will be located as losses of nuclease cleavages, and mRNA conformation will be assessed based on specificity of cleavages. These experiments will identify bona fide mRNA binding sites for FMRP and their structural environment. Specific Aim 2 will characterize mRNA binding sites for FMRP biochemically and structurally. Representative mRNAs will be tested for binding to various domains of FMRP. Identified RNA-protein complexes will be co-crystallized and their structures determined by X-ray crystallography. Together, results will define structural and sequence elements required for interactions with FMRP. Complemented by in silico characterization of the sites, these data will provide 'three-dimensional' RNA binding signatures for FMRP. Thus the proposed study will define the principles of mRNA-FMRP recognition and provide insights on the mechanism of FMRP-dependent translational inhibition. The proposal is relevant to public health since it addresses the molecular basis of the most common cause of mental retardation and related disorders. Understanding FMRP function will advance searches for novel therapeutic interventions against this incurable disease. Thus, the proposed research is relevant to the NIH mission to expand the knowledge base in medical sciences to ensure capability to prevent and cure diseases.

描述（由申请人提供）：mRNA结合蛋白功能的缺陷是人类病理的广泛。脆弱的智力低下蛋白（FMRP）是这些mRNA结合蛋白之一，涉及mRNA的运输，储存和控制。其生物合成的变化会导致几种发育和认知缺陷，包括早产卵巢衰竭和脆弱的X综合征（FXS）。 FXS是最常见的智障形式，是自闭症的主要单基因原因。该综合征与FMRP基因的转录沉默或FMRP的RNA结合结构域中的突变有关。在分子水平上，FXS可能是由FMRP介导的转化控制失调引起的，导致突触功能改变和其他异常。尽管通过各种方法鉴定了许多与FMRP相关的mRNA，但FMRP识别mRNA的基础仍然尚不清楚。该提案的目的是恢复旨在阐明FMRP识别mRNA识别原理的试点研究项目。假设是FMRP识别具有序列和结构特异性结合决定因素的mRNA靶标。为了检验这一假设，建议使用新方法鉴定FMRP结合位点，并以生化和生物物理方式表征FMRP-RNA相互作用。具体目标1致力于开发全基因组RNA脚印，该印刷具有互补特异性的构象特异性核酸酶，以鉴定FMRP结合位点并探测其构象。在不存在和存在FMRP的情况下，将通过小鼠mRNA的核酸酶切割在体外测试mRNA结构。切割的mRNA片段将转换为cDNA并测序。 FMRP结合位点将被视为核酸酶裂解的损失，将根据裂解的特异性评估mRNA构象。这些实验将确定FMRP及其结构环境的真正的mRNA结合位点。特定的目标2将在生化和结构上表征FMRP的mRNA结合位点。代表性mRNA将测试与FMRP的各个领域的结合。鉴定的RNA蛋白质复合物将被共结晶，并由X射线晶体学确定其结构。总之，结果将定义与FMRP相互作用所需的结构和序列元素。在对位点的硅表征中的补充，这些数据将为FMRP提供“三维” RNA结合特征。因此，拟议的研究将定义mRNA-FMRP识别的原理，并提供有关FMRP依赖性翻译抑制机制的见解。该提案与公共卫生有关，因为它解决了智力低下和相关疾病最常见原因的分子基础。了解FMRP功能将推动针对这种无法治愈的疾病进行新的治疗干预措施的搜索。因此，拟议的研究与NIH的使命有关，以扩大医学科学的知识库，以确保能力预防和治愈疾病。