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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）：脆性X型智力发育迟滞综合征是遗传性智力发育迟滞最常见的一种，男性4000例中有1例，女性8000例中有1例。这种综合征是由一种正常细胞蛋白的丢失引起的，这种蛋白被称为脆性X智力迟钝蛋白（FMRP）。尽管在过去的15年中进行了广泛的研究，但FMRP缺失与脆性X综合征表型之间的关系仍未完全了解。FMRP是一种RNA结合蛋白，被认为参与特定信使RNA （mRNA）靶点的转运和翻译调节。生化研究已经确定，FMRP利用其精氨酸-甘氨酸-甘氨酸（RGG）盒以高亲和力结合有可能形成G四重结构的RNA序列。本项目重点研究一种具有较强特异性的mRNA