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

项目摘要 脆性X智力低下综合征是遗传性智力损害的最常见形式，影响1 在4000名男性和1名6000名女性中。这种综合征是由一种正常的细胞蛋白质丢失引起的，这种蛋白质名为 脆性X智力低下蛋白(FMRP)。FMRP是一种RNA结合蛋白，参与转运和 特定信使RNA(信使RNA)靶标的翻译调节。FMRP发挥作用的机制 翻译调节功能尚不清楚，但已提出蛋白质协同工作。 通过microRNA(MiRNA)途径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，能够处理前体microRNAs(前miRNAs)， 有可能参与成熟的miRNA的生物发生。这一提议将成为这部小说的特点 FMRP功能，有以下具体目标： 目的I.鉴定与其核酸酶活性有关的FMRP结构域(S)。我们假设 FMRP核酸酶活性位于他的K同源结构域之一，为了验证这一假设，我们将产生 几个缺少一个或多个这些结构域的FMRP构建体。我们确定了磷酸化的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于它们的不同 二聚化性质。最后，我们还将测试FMRP是否与FXR1P和FXR2P类似，它们与 FMRP的KH0、Kh1和Kh2结构域也具有核酸酶活性。 目的II.FMRP核酸酶活性的生化特性。我们将确定前miRNA是否 FMRP及其磷酸化的模拟物可以将前miRNAs切割成成熟的miRNAs。此外，我们将确定 如果FMRP有额外的核酸酶活性底物，如RNA完美双链、RNA单链、RNA G四链、DNA-RNA杂合双链、DNA双链。最后，我们将表征FMRP的动力学 核酸酶。 目的III.研究FMRP与SARS-CoV-2 3‘-UTR基因组的相互作用及其潜力 其核酸酶活性在该病毒系统中所起的作用。类似于它所展示的制约因素的作用 在寨卡病毒感染中，FMRP可能在严重急性呼吸道疾病中发挥作用 综合症冠状病毒2(SARS-CoV-2)感染，是导致当前新冠肺炎大流行的病毒。我们 将测试FMRP是否切割SARS-CoV-2RNA基因组3‘-非翻译区内的各种茎环 它的核酸酶功能要么潜在地产生病毒miRNAs，要么在抗病毒宿主反应中发挥作用。

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.