RNA Targets for Fragile X Mental Retardation Protein

摘要 信使核糖核酸结合蛋白在神经系统和缺陷的发育和功能中起着关键作用。 在这些蛋白质的功能中，存在着广泛的神经病理基础。脆弱的X心理 迟缓蛋白(FMRP)是疾病相关RNA结合蛋白的一种范例，因为它具有重要的 对大脑的发育和活动的贡献及其在几种人类疾病中的核心作用 影响着数十万人。转录沉默或蛋白质突变导致的FMRP丢失 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性原因 目前缺乏有效的医疗救治。在分子水平上，缺乏功能性FMRP结果 在通常由FMRP介导的翻译抑制所抑制的夸大的蛋白质生物合成中 所选的mRNAs。之前的研究报告了FMRP目标的数据集大多相互冲突，尽管 尽管FMRP对mRNA的选择具有重要意义，但其作用机制尚不清楚。这种缺乏确定性的 对FMRP-RNA识别原理的了解限制了对FXS的理解和开发 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，FMRP的RNA结合并不是真正的混杂。目标是 这项建议的目的是确定人FMRP的特定RNA靶点，并了解其分子原理 FMRP-RNA识别的结果。假设FMRP的RNA结合域识别RNA序列- 具体地说，这些RNA基序组合决定了与天然RNA的结合。为了测试这一点 假设，FMRP结合位点将使用一种新的生化方法和结构研究来确定。 具体目标1致力于鉴定与分离的KH结构域特异结合的短RNA序列 通过使用一种新的将RNA捕获实验与NEXT相结合的“自下而上”方法来研究FMRP 世代排序。具体目标2将描述FMRP的分子特征，这些分子特征对于 用生化方法和X射线结晶学方法进行特异的RNA结合。具体目标3将致力于 开发RNA特异性改变的突变型FMRP蛋白，以研究FMRP的各种功能。加在一起，这些 结果将确定与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蛋白来询问动物的各种FMRP功能 FXS的型号。该提案与公共卫生和美国国立卫生研究院的任务高度相关，因为它将提供 对RNA识别和FMRP介导的翻译抑制机制、活性的认识 与FXS、自闭症和其他障碍的发展有关。了解FMRP如何运作将 针对FXS和相关疾病的新的治疗干预措施的先进研究。

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.