Functional studies of the Fragile X Mental Retardation Protein: switching from re

脆性 X 智力迟钝蛋白的功能研究：从 re 转变

基本信息

批准号：
7934339
负责人：
MIHAELA R MIHAILESCU
金额：
$ 12.4万
依托单位：
DUQUESNE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7934339
关键词：
5&apos Untranslated Regions Affect Affinity Alternative Splicing Arginine Bacteria Binding Biochemical Boxing Chemicals Event Exons FMR1 Gene Female Fragile X Mental Retardation Protein Fragile X Syndrome G-Quartets Gene Expression Gene Expression Regulation Glycine In Vitro Inherited Investigation KH Domain Knowledge Laboratories Literature Mental Retardation Messenger RNA Methylation Modeling Modification Molecular N-terminal Names Neurotransmitters Phenotype Phosphorylation Play Polyribosomes Post-Translational Protein Processing Property Protein Dephosphorylation Protein Isoforms Protein-Arginine N-Methyltransferase Proteins Public Health RNA RNA Binding RNA Recognition Motif RNA Sequences RNA-Binding Proteins Recombinants Regulation Reporter Genes Research Role SUI1 gene Signal Transduction Structure Syndrome Tertiary Protein Structure Testing Translations Variant Work base comparative design in vivo male microtubule-associated protein 1B protein function response translation assay

项目摘要

DESCRIPTION (provided by applicant): Fragile X mental retardation syndrome is the most common form of inherited mental retardation, affecting ~1 in 4000 males and ~ 1 in 8000 females. The syndrome is caused by the loss of a normal cellular protein, named the fragile X mental retardation protein (FMRP). Despite extensive research in the past fifteen years, the relationship between the absence of FMRP and the phenotype of the fragile X syndrome is still not fully understood. FMRP is an RNA binding protein believed to be involved in the transport and translation regulation of specific messenger RNA (mRNA) targets. Biochemical studies have determined that FMRP uses its arginine-glycine-glycine (RGG) box to bind with high affinity to RNA sequences that have the potential to form G quadruplex structures. This project focuses on the study of one such mRNA for which there is strong evidence in the literature that it is a relevant in vivo FMRP target, namely the microtubule associated protein 1B (MAP1B) mRNA. This proposal has the following specific aims: 1. Investigation of a regulatory switch for FMRP function: from translation repressor to translation activator of G quadruplex forming mRNA. We proposed a model according to which the variation of the FMRP concentration in response to a neurotransmitter stimulation event acts as a switch for the protein function from repressor to activator of translation of its G quadruplex forming mRNA targets. This model will be tested by using recombinant FMRP ISO1 in an in vitro translation assay of a reporter gene that contains the MAP1B RNA G quadruplex structure in its 5'-untranslated region (UTR). 2. Investigation of the role played by protein post-translational modifications (phosphorylation and arginine methylation) in modulating the FMRP translation regulator function. We will phosphorylate and arginine methylate FMRP ISO1 that has been expressed in bacteria devoid of posttranslational modifications and will quantify its binding properties to the G quadruplex forming MAP1B RNA, as well as its ability to regulate the translation of a reporter gene that has this G quadruplex structure in its 5'-UTR. 3. Characterization of different FMRP isoforms interactions with the G quadruplex forming MAP1B mRNA. We will perform a comparative analysis of the FMRP isoforms ISO1, ISO2 and ISO3 interactions with the G quadruplex forming MAP1B RNA to determine at the molecular level to what extent naturally occurring sequence modifications near the RGG box (occurring in the ISO2 and ISO3 isoforms) affect the FMRP interactions with its specific RNA targets. PUBLIC HEALTH REVELANCE: This is a detailed study of the FMRP-RNA interactions at the molecular level, whose results will contribute to our understanding of the mechanisms by which FMRP achieves its function of translation regulator. The identification of such mechanisms and of the regulatory signals that modulate them could in turn facilitate the design and analysis of synthetic chemical compounds that mimic the protein function. In addition, this study of the FMRP interactions with the G quadruplex forming MAP1B mRNA is valuable beyond the fragile X syndrome context, in that will provide information about the role of the RNA G quadruplex structure in the regulation of gene expression at the translational level.

描述（由申请人提供）：脆弱的X心理迟缓综合征是遗传性智力低下的最常见形式，影响了4000名男性中的约1，而8000名女性中有〜1。该综合征是由正常细胞蛋白的丧失引起的，该蛋白质称为脆弱的X智障蛋白（FMRP）。尽管在过去的十五年中进行了广泛的研究，但仍未完全了解FMRP的缺乏与脆弱X综合征的表型之间的关系。 FMRP是一种RNA结合蛋白，据信与特定信使RNA（mRNA）靶标的转运和翻译调节有关。生化研究已经确定FMRP使用其精氨酸 - 甘氨酸 - 甘氨酸（RGG）盒与有可能形成G四链体结构的RNA序列的高亲和力结合。该项目的重点是对有很强的一个这样的mRNA的研究文献中的证据表明它是体内FMRP靶标相关的，即微管相关蛋白1b（MAP1B）mRNA。该建议具有以下具体目标： 1。调查FMRP功能的调节开关：从翻译阻遏物到G四链体形成mRNA的翻译激活剂。我们提出了一个模型，根据该模型，FMRP浓度对神经递质刺激事件的响应变化可作为蛋白质函数从抑制剂到其G四倍体形成mRNA靶标转换的蛋白质函数的开关。该模型将通过在其5'-非翻译区域（UTR）中包含MAP1B RNA G四链体结构的报告基因的体外翻译测定中使用重组FMRP ISO1进行测试。 2。研究蛋白质后翻译后修饰（磷酸化和精氨酸甲基化）在调节FMRP翻译调节仪函数中所起的作用。我们将磷酸化和精氨酸甲基酸FMRP ISO1在没有翻译后修饰的细菌中表达，并将量化其与G Quadruplex形成MAP1B RNA的结合特性，以及其能够调节该Quadruplex结构在其5'5'-usrr中的转化基因的能力。 3。与G四链体形成MAP1B mRNA的不同FMRP同工型相互作用的表征。我们将对FMRP同工型ISO1，ISO2和ISO3与G Quadruplex形成MAP1B RNA的相互作用进行比较分析，以在分子级确定RGG盒附近自然发生的序列修饰的程度（在ISO2和ISO3 ISOForms中发生自然发生的序列修饰的程度）与FMRP的相互作用发生在ISO2和ISO3 ISOForms中。公共卫生启示：这是对分子水平上FMRP-RNA相互作用的详细研究，其结果将有助于我们理解FMRP实现其翻译调节剂功能的机制。调节它们的这种机制和调节信号的鉴定可以促进模仿蛋白质功能的合成化合物的设计和分析。此外，对FMRP与G四链体形成MAP1B mRNA的相互作用的研究超出了脆弱的X综合征环境，因为它将提供有关RNA G四倍体结构在翻译水平上基因表达中的作用的信息。