G quartet RNA-FMRP interactions in Fragile X syndrome

描述（由申请人提供）：该资助重点研究脆性X智力迟钝蛋白（FMRP）与G四重奏形成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最常见的一种形式，每4000名男性和8000名女性中分别有1名和1名患者。该综合征是由正常细胞蛋白（FMRP）的缺失引起的，该蛋白被认为是特定信使RNA （mRNA）的翻译抑制因子。尽管FMRP蛋白的体内RNA靶点仍然难以捉摸，但据报道，FMRP与富含鸟嘌呤含量的RNA序列高亲和力结合，这些RNA序列折叠成G四重奏结构。鉴于FMRP RNA结合活性对其功能的重要性，本资助的具体目标之一是研究FMRP RNA识别的分子基础。含有RNA的g -四重奏是通过序列特异性识别，还是通过结构特异性机制识别，还是两者都识别？FMRP是稳定还是破坏这些结构？FMRP利用其富含精氨酸-甘氨酸的结构域（RGG box）与G四重奏形成的RNA结合，该结构域存在于许多其他RNA结合蛋白中。该建议的另一个目的是通过分析来自其他RNA结合蛋白的2种不同RGG盒与这些RNA序列的相互作用，确定G四重奏结构被该结构域识别的特异性水平。该提案的第三个目的是确定蛋白质精氨酸甲基化是否在调节FMRP-RNA相互作用中起作用，这是一种常见的翻译后修饰，涉及RGG盒内精氨酸残基的甲基化。这些问题的答案将有助于我们识别FMRP的体内靶点，从而努力理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被用来生产RNA和蛋白质，生物物理技术，如核磁共振光谱，荧光光谱，紫外-可见光谱将被用来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.