G quartet RNA-FMRP interactions in Fragile X syndrome

描述（由申请人提供）：该基金的重点是研究脆性X智力低下蛋白（FMRP）与G四联体形成RNA靶标的相互作用。脆性X染色体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。该综合征是由正常细胞蛋白（FMRP）的丢失引起的，FMRP被认为是特异性信使RNA（mRNA）的翻译阻遏物。尽管该蛋白的体内RNA靶标仍然难以捉摸，但据报道，FMRP以高亲和力结合富含鸟嘌呤含量的RNA序列，其折叠成G四联体结构。鉴于FMRP RNA结合活性对其功能的重要性，该资助的具体目标之一是研究FMRP识别RNA的分子基础。含有G-四联体的RNA是通过序列特异性、结构特异性机制或两者来识别的吗？FMRP是稳定还是破坏了这些结构？FMRP使用其富含甘氨酸的结构域（RGG盒）结合G四联体形成RNA，该结构域在许多其他RNA结合蛋白中发现。该提案的另一个目的是通过分析衍生自其他RNA结合蛋白的2种不同RGG盒与这些RNA序列的相互作用来确定该结构域识别G四联体结构的特异性水平。该提案的第三个目的是确定蛋白质精氨酸甲基化，一种常见的翻译后修饰，涉及RGG盒内精氨酸残基的甲基化，是否在调节FMRP-RNA相互作用中起作用。对这些问题的回答将有助于我们识别FMRP体内靶点的能力，以了解蛋白质缺失与脆性X综合征表型之间的联系。为了实现这些目标，分子生物学和生物化学方法将用于产生RNA和蛋白质，生物物理技术如NMR光谱，荧光光谱，UV-Vis光谱将用于研究这些生物分子及其相互作用。

DESCRIPTION (provided by applicant): This grant focuses on the investigation of the fragile X mental retardation protein (FMRP) interactions with G quartet forming RNA target(s). Fragile X 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 (FMRP), which is thought to act as a translational repressor of specific messenger RNA (mRNA). Although the in vivo RNA targets of the protein remain elusive, it has been reported that FMRP binds with high affinity to RNA sequences rich in guanine content, which fold into G quartet structures. Given the importance of the FMRP RNA binding activity for its function, one of the specific aims of this grant is to investigate the molecular basis of RNA recognition by FMRP. Is the G-quartet containing RNA recognized via a sequence specific, via a structure specific mechanism, or both? Is FMRP stabilizing or destabilizing these structures? FMRP binds the G quartet forming RNA using its arginine-glycine rich domain (RGG box), a domain found in many other RNA binding proteins. Another aim of the proposal is to determine the level of specificity with which the G quartet structures are recognized by this domain, by analyzing the interactions of 2 different RGG boxes derived from other RNA binding proteins with these RNA sequences. The third aim of the proposal is to determine if protein arginine methylation, a common posttranslational modification involving the methylation of arginine residues within the RGG box, plays a role in modulating the FMRP-RNA interactions. Answers to such questions will contribute to our ability to identify the in vivo targets of FMRP, in an effort to understand the link between the absence of the protein and the phenotype of the fragile X syndrome. To accomplish these goals, molecular biology and biochemistry methods will be used to produce the RNA and protein and biophysical techniques such as NMR spectroscopy, fluorescence spectroscopy, UV-Vis spectroscopy will be employed to study these biomolecules and their interactions.