RNA-protein interactions in Fragile X syndrome

描述（由申请人提供）：mRNA结合蛋白功能缺陷是多种人类病理学的基础。脆性智力迟钝蛋白（FMRP）是这些mRNA结合蛋白中的一种，参与mRNA的运输、储存和控制;其生物合成的变化导致几种发育和认知缺陷，包括卵巢早衰和脆性X综合征（FXS）。FXS是智力残疾的最常见遗传形式，也是自闭症的主要单基因原因。该综合征与FMRP基因的转录沉默或FMRP的RNA结合结构域的突变有关。在分子水平上，FXS可能是由神经元中FMRP介导的翻译控制失调引起的，导致突触功能改变和其他异常。尽管通过各种方法鉴定了许多FMRP相关的mRNA，但FMRP识别mRNA的基础仍然不清楚。该提案的目的是恢复专注于阐明FMRP识别mRNA的原则的试点研究项目。假设是FMRP识别具有序列和结构特异性结合决定簇的mRNA靶。为了验证这一假设，建议使用新的方法鉴定FMRP结合位点，并从生物化学和生物药理学上表征FMRP与RNA的相互作用。Specific Aim 1致力于开发全基因组RNA足迹，其实现具有互补特异性的构象特异性核酸酶，以识别FMRP结合位点并探测其构象。在不存在和存在FMRP的情况下，通过核酸酶切割总小鼠mRNA，在体外检测mRNA结构。将切割的mRNA片段转化为cDNA并测序。FMRP结合位点将被定位为核酸酶切割的损失，并且mRNA构象将基于切割的特异性来评估。这些实验将鉴定FMRP的真正mRNA结合位点及其结构环境。特异性目的2将在生物化学和结构上表征FMRP的mRNA结合位点。将测试代表性mRNA与FMRP的各种结构域的结合。将鉴定的RNA-蛋白质复合物共结晶，并通过X射线晶体学确定其结构。总之，结果将定义与FMRP相互作用所需的结构和序列元素。通过对这些位点的计算机表征，这些数据将为FMRP提供“三维”RNA结合特征。因此，拟议的研究将定义mRNA-FMRP识别的原则，并提供对FMRP依赖的翻译抑制机制的见解。该提案与公共卫生有关，因为它涉及智力迟钝和相关疾病最常见原因的分子基础。了解FMRP功能将促进对这种不治之症的新型治疗干预的研究。因此，拟议的研究与NIH的使命有关，即扩大医学科学的知识基础，以确保预防和治疗疾病的能力。

DESCRIPTION (provided by applicant): Defects in the function of mRNA-binding proteins underlie a broad spectrum of human pathologies. Fragile Mental Retardation Protein (FMRP) is one of these mRNA-binding proteins, involved in transport, storage and control of mRNA; changes in its biosynthesis cause several developmental and cognitive deficiencies including premature ovarian failure and fragile X syndrome (FXS). FXS is the most common inherited form of intellectual disability and a predominant monogenic cause of autism. The syndrome is associated with transcriptional silencing of the FMRP gene or a mutation in an RNA-binding domain of FMRP. On the molecular level, FXS is likely caused by dysregulation of FMRP-mediated translational control in neurons leading to altered synaptic function and other abnormalities. Despite identification of many FMRP-associated mRNAs by various approaches, the basis for mRNA recognition by FMRP is still not understood. The objective of this proposal is to restore the pilot research project focused on elucidating the principles of mRNA recognition by FMRP. The hypothesis is that FMRP recognizes mRNA targets that bear both sequence and structure-specific binding determinants. To test this hypothesis, it is proposed to identify FMRP binding sites using novel methodology and characterize FMRP-RNA interactions biochemically and biophysically. Specific Aim 1 is devoted to development of genome-wide RNA foot printing that implements conformation-specific nucleases with complementary specificities to identify FMRP-binding sites and probe their conformation. The mRNA structure will be tested in vitro by nuclease cleavage of total mouse mRNA in the absence and presence of FMRP. The cleaved mRNA fragments will be converted to cDNA and sequenced. FMRP binding sites will be located as losses of nuclease cleavages, and mRNA conformation will be assessed based on specificity of cleavages. These experiments will identify bona fide mRNA binding sites for FMRP and their structural environment. Specific Aim 2 will characterize mRNA binding sites for FMRP biochemically and structurally. Representative mRNAs will be tested for binding to various domains of FMRP. Identified RNA-protein complexes will be co-crystallized and their structures determined by X-ray crystallography. Together, results will define structural and sequence elements required for interactions with FMRP. Complemented by in silico characterization of the sites, these data will provide 'three-dimensional' RNA binding signatures for FMRP. Thus the proposed study will define the principles of mRNA-FMRP recognition and provide insights on the mechanism of FMRP-dependent translational inhibition. The proposal is relevant to public health since it addresses the molecular basis of the most common cause of mental retardation and related disorders. Understanding FMRP function will advance searches for novel therapeutic interventions against this incurable disease. Thus, the proposed research is relevant to the NIH mission to expand the knowledge base in medical sciences to ensure capability to prevent and cure diseases.