RNA-protein interactions in Fragile X syndrome

描述(由申请人提供)：信使核糖核酸结合蛋白的功能缺陷是人类广泛的病理基础。脆性智力低下蛋白(FMRP)是一种与mRNA结合的蛋白，参与了mRNA的运输、储存和调控，其生物合成的改变会导致多种发育和认知缺陷，包括卵巢早衰和脆性X综合征(FXS)。FXS是最常见的遗传性智力残疾形式，也是导致自闭症的主要单基因原因。该综合征与FMRP基因转录沉默或FMRP的RNA结合域突变有关。在分子水平上，FXS可能是由于FMRP介导的神经元翻译控制失调导致突触功能改变和其他异常所致。尽管通过不同的方法鉴定了许多FMRP相关的mRNAs，但FMRP识别mRNAs的基础仍然不清楚。这项建议的目的是恢复以阐明FMRP识别信使核糖核酸的原理为重点的试点研究项目。假设FMRP识别同时具有序列和结构特异性结合决定因素的mRNA靶标。为了验证这一假设，建议使用新的方法确定FMRP结合部位，并从生化和生物物理角度表征FMRP与RNA的相互作用。具体目标1致力于开发全基因组RNA足迹印迹，实现具有互补特异性的构象特异核酸酶，以识别FMRP结合位点并探测其构象。在没有FMRP和存在FMRP的情况下，通过对小鼠总mRNA的核酸酶裂解，在体外测试mRNA的结构。将切割后的mRNA片段转化为cDNA并测序。FMRP结合位点将被定位为核酸酶裂解的丢失，而mRNA的构象将基于裂解的特异性进行评估。这些实验将确定FMRP的真正mRNA结合位点及其结构环境。特定目的2将从生化和结构上表征FMRP的mRNA结合位点。将测试具有代表性的mRNAs与FMRP的不同结构域的结合。已确定的RNA-蛋白质复合体将被共结晶，其结构将通过X射线结晶学确定。结合起来，结果将定义与FMRP相互作用所需的结构和序列元素。在位点的电子表征的补充下，这些数据将为FMRP提供“三维”的RNA结合签名。因此，本研究将确定mRNA-FMRP识别的原理，并为FMRP依赖的翻译抑制机制提供见解。这项提案与公共健康相关，因为它涉及导致精神发育迟滞和相关疾病的最常见原因的分子基础。了解FMRP的功能将促进对这种不治之症的新的治疗干预措施的探索。因此，这项拟议的研究与美国国立卫生研究院扩大医学知识库以确保预防和治疗疾病的能力的使命有关。

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.