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

描述（由申请人提供）：脆性X染色体智力低下综合征是遗传性智力低下的最常见形式，影响约1/4000男性和约1/8000女性。这种综合征是由一种正常细胞蛋白质的丢失引起的，这种蛋白质被称为脆性X智力低下蛋白（FMRP）。尽管在过去的15年中进行了广泛的研究，但FMRP的缺失与脆性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，被认为参与特异性信使RNA（mRNA）靶标的转运和翻译调节。生物化学研究已经确定FMRP使用其甘氨酸-甘氨酸-甘氨酸（RGG）盒以高亲和力结合具有形成G四链体结构的潜力的RNA序列。该项目的重点是研究一种这样的mRNA， 文献中的证据表明，它是一个相关的体内FMRP靶点，即微管相关蛋白1B（MAP 1B）mRNA。这项建议的具体目标如下： 1. FMRP功能调控开关的研究：从G四链体形成mRNA的翻译阻遏物到翻译激活物。我们提出了一个模型，根据该模型，响应神经递质刺激事件的FMRP浓度变化充当蛋白质功能从G四链体形成mRNA靶点翻译的抑制剂到激活剂的开关。将在报告基因的体外翻译试验中使用重组FMRP ISO 1检测该模型，该报告基因在其5 '-非翻译区（UTR）中含有MAP 1B RNA G四链体结构。 2.研究蛋白质翻译后修饰（磷酸化和精氨酸甲基化）在调节FMRP翻译调节因子功能中的作用。我们将磷酸化和精氨酸甲基化FMRP ISO 1，其已在没有翻译后修饰的细菌中表达，并将量化其与G四链体形成MAP 1B RNA的结合特性，以及其调节在其5 '-UTR中具有该G四链体结构的报告基因的翻译的能力。 3.不同FMRP同种型与G四链体形成MAP 1B mRNA相互作用的表征。我们将对FMRP亚型ISO 1、ISO 2和ISO 3与G四链体形成的MAP 1B RNA的相互作用进行比较分析，以确定在分子水平上，RGG盒附近天然存在的序列修饰（发生在ISO 2和ISO 3亚型中）在多大程度上影响FMRP与其特异性RNA靶点的相互作用。 公共卫生部门：本文从分子水平上对FMRP与RNA的相互作用进行了详细的研究，其结果将有助于我们理解FMRP发挥其翻译调节功能的机制。这些机制和调节它们的调节信号的识别反过来可以促进模拟蛋白质功能的合成化合物的设计和分析。此外，这项研究的FMRP与G四链体形成MAP 1B mRNA的相互作用是有价值的脆性X综合征的背景下，在这将提供信息的作用RNA G四链体结构在基因表达的调控在翻译水平。

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.