Biochemical characterization of a novel Fragile X Mental Retardation Protein nuclease function

新型脆性 X 智力迟钝蛋白核酸酶功能的生化表征

• 批准号: 10359289
• 负责人: MIHAELA R MIHAILESCU
• 金额: $ 41.4万
• 依托单位: DUQUESNE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359289
• 关键词: 2019-nCoV; 3' Untranslated Regions; Active Sites; Affect; Affinity; Amino Acids; Antiviral Agents; Arginine; Binding; Biochemical; Biogenesis; Biological; Biological Assay; Biological Process; C-terminal; COVID-19 pandemic; Cleaved cell; Complex; DNA; Dependence; Development; Dimerization; FMR1; FXR1 gene; FXR2 gene; Female; Fluorescence Spectroscopy; Fragile X Syndrome; G-Quartets; Genome; Glycine; Guide RNA; Homodimerization; Hybrids; Immune response; Individual; Inherited; Investigation; Ions; KH Domain; Kinetics; Literature; Mass Spectrum Analysis; Messenger RNA; MicroRNAs; Modeling; N-terminal; Names; Northern Blotting; Nucleotides; Pathway interactions; Phosphorylation; Play; Point Mutation; Process; Property; Protein Biosynthesis; Proteins; Psyche structure; RNA; RNA Interference; RNA Recognition Motif; RNA Viruses; RNA-Binding Proteins; Recombinants; Regulation; Reporting; Role; SARS-CoV-2 antiviral; SARS-CoV-2 genome; SARS-CoV-2 infection; Specificity; Structure; Synapses; Syndrome; System; Tertiary Protein Structure; Testing; Translations; Untranslated RNA; Viral; Virus; Work; ZIKV infection; Zika Virus; genomic RNA; male; novel; nuclease; paralogous gene; protein function; quadruplex DNA; response; stem

Project Summary Fragile X mental retardation syndrome is the most common form of inherited mental impairement, affecting ~ 1 in 4000 males and ~ 1 in 6000 females. The syndrome is caused by the loss of a normal cellular protein, named the fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein involved in the transport and translation regulation of specific messenger RNA (mRNA) targets. The mechanisms by which FMRP exerts its translation regulator function are not known, however it has been proposed that the protein works in conjunction with the microRNA (miRNA) pathway to regulate local protein synthesis in response to synaptic input. We have determined that FMRP has nuclease activity, being able to process precursor microRNAs (pre-miRNAs), potentially being involved in the mature miRNA biogenesis. This proposal, which will characterize this novel FMRP function, has the following specific aims: AIM I. Identification of the FMRP domain(s) responsible for its nuclease activity. We hypothesized that the FMRP nuclease activity resides in one of his K homology domains and to test this hypothesis we will produce several FMRP constructs lacking one or more of these domains. We determined that phosphorylated FMRP has higher nuclease efficiency than the unphosphorylated FMRP and we will test if this is caused by their different dimerization properties. Finally, we will also test if the FMRP paralogs, FXR1P and FXR2P, which share with FMRP the KH0, KH1 and KH2 domains, also have nuclease activity. AIM II. Biochemical characterization of the FMRP nuclease activity. We will determine if the pre-miRNA FMRP and its phosphorylated mimic can cleave pre-miRNAs into mature miRNAs. Additionally, we will determine if FMRP has additional substrates for its nuclease activity such as RNA perfect duplex, RNA single strand, RNA G quadruplex, DNA-RNA hybrid duplex, DNA duplex. Finally, we will characterize the kinetics of the FMRP nuclease. AIM III. Investigation of the FMRP interactions with the SARS-CoV-2 3’-UTR genome and of the potential role played by its nuclease activity in this viral system. Similar to its demonstrated role of restrictive factor in ZIKA virus infection, it has been proposed that FMRP might play a role in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the virus responsible for the current COVID-19 pandemic. We will test if FMRP cleaves various stem-loops within the SARS-CoV-2 RNA genomic 3’-untranslated region using its nuclease function either to potentially yield viral miRNAs or functioning in the antiviral host response.

项目摘要 脆性X染色体智力低下综合征是遗传性智力障碍的最常见形式， 4000例男性和~1/6000例女性。该综合征是由一种正常细胞蛋白质的丢失引起的， 脆性X智力低下蛋白（FMRP）FMRP是一种参与转运的RNA结合蛋白， 特定信使RNA（mRNA）靶点的翻译调节。FMRP发挥其作用的机制 翻译调节因子的功能尚不清楚，但是已经提出该蛋白质与 与microRNA（miRNA）通路调节局部蛋白质合成，以响应突触输入。我们有 确定FMRP具有核酸酶活性，能够加工前体microRNA（pre-miRNAs）， 可能参与成熟miRNA的生物发生。这一提议将成为这部小说的特色 FMRP功能具有以下具体目标： AIM岛鉴定负责其核酸酶活性的FMRP结构域。我们假设 FMRP核酸酶活性存在于他的K同源结构域之一，为了验证这一假设，我们将产生 几种缺乏一个或多个这些结构域的FMRP构建体。我们确定磷酸化的FMRP 比未磷酸化的FMRP更高的核酸酶效率，我们将测试这是否是由它们的不同 二聚特性。最后，我们还将测试FMRP的旁系同源物FXR1P和FXR2P是否与 FMRP的KH 0、KH 1和KH 2结构域也具有核酸酶活性。 AIM II. FMRP核酸酶活性的生物化学表征。我们将确定前体miRNA FMRP及其磷酸化模拟物可以将pre-miRNA切割成成熟的miRNA。此外，我们将确定 如果FMRP具有用于其核酸酶活性的其它底物，例如RNA完全双链体、RNA单链、RNA G四链体、DNA-RNA杂交双链体、DNA双链体。最后，我们将表征FMRP的动力学 核酸酶。 AIM III.研究FMRP与SARS-CoV-2 3'-UTR基因组的相互作用以及FMRP与SARS-CoV-2 3'-UTR基因组的潜在相互作用。 它的核酸酶活性在该病毒系统中发挥的作用。与其所显示的限制性因素作用相似 在ZIKA病毒感染中，已经提出FMRP可能在严重急性呼吸道感染中起作用， 综合征冠状病毒2（SARS-CoV-2）感染，该病毒导致当前的COVID-19大流行。我们 将测试FMRP是否切割SARS-CoV-2 RNA基因组3 '非翻译区内的各种茎环， 其核酸酶的功能是潜在地产生病毒miRNA或在抗病毒宿主应答中起作用。