RNA-protein interactions in Fragile X syndrome

描述（由申请人提供）：mrna结合蛋白的功能缺陷是广泛的人类病理的基础。脆性智力迟钝蛋白（Fragile Mental retardp Protein， FMRP）是一类mRNA结合蛋白，参与mRNA的转运、储存和调控；其生物合成的改变导致多种发育和认知缺陷，包括卵巢早衰和脆性X染色体综合征（FXS）。FXS是智力残疾最常见的遗传形式，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 rna结合域的突变有关。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导致突触功能改变和其他异常引起的。尽管通过各种方法鉴定了许多与FMRP相关的mRNA，但FMRP识别mRNA的基础仍然不清楚。本提案的目的是恢复试点研究项目的重点是阐明mRNA识别原理的FMRP。假设是FMRP识别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，我们建议使用新的方法确定FMRP结合位点，并从生物化学和生物物理角度表征FMRP- rna相互作用。Specific Aim 1致力于开发全基因组RNA足迹，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。mRNA的结构将在体外通过核酸酶切割小鼠总mRNA，在没有和存在FMRP的情况下进行测试。被切割的mRNA片段将被转化为cDNA并测序。FMRP结合位点将定位为核酸酶裂解的损失，并根据裂解的特异性评估mRNA的构象。这些实验将确定FMRP真正的mRNA结合位点及其结构环境。特异性Aim 2将从生物化学和结构上表征FMRP的mRNA结合位点。将测试具有代表性的mrna与FMRP的不同结构域的结合。鉴定出的rna -蛋白复合物将被共结晶，并用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，本研究将明确mRNA-FMRP识别的原理，并对fmrp依赖性翻译抑制的机制提供见解。该建议与公共卫生有关，因为它涉及精神发育迟滞和相关疾病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库，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.