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

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），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。我们已经确定FMRP具有核酸酶 活性，能够加工前体microRNA（pre-miRNAs），可能参与成熟的 miRNA生物发生父母的建议，这将表征这种新的FMRP功能，有以下几点 具体目标： AIM岛鉴定负责其核酸酶活性的FMRP结构域。在这一目标下，我们将 确定：（i）FMRP核酸酶活性是否存在于其KH结构域之一中，并鉴定活性位点残基 （ii）如果FMRP S500 D与FMRP S500 D相比具有更高的核酸酶活性， 由于不同的二聚化特性，和（iii）如果旁系同源物FXR 1 P和FXR 2 P， 与FMRP共享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。在这一具体目标下，我们将： 确定FMRP和FMRP S500 D是否将pre-miRNA切割成成熟的miRNA;（ii）确定它们是否具有 其他底物如RNA完全双链体、RNA单链、RNA G四链体、DNA-RNA杂合体 双链体，DNA双链体;（iii）表征其核酸酶活性的动力学。将进行类似的实验 以测试FMRP旁系同源物FXR 1 P和FXR 2 P的核酸酶活性。 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。在此目标下，我们将调查FMRP和FMRP S500 D以及FXR 1 P和FXR 2 P与SARS-CoV-2 3 '-UTR基因组相互作用，切割保守的茎- 环，以潜在地产生病毒miRNA或在抗病毒宿主反应中起作用。 荧光光谱法将用于R15母提案的所有三个具体目标， Fluorolog-QM-75-11-C荧光分光光度计和附件是成功的建议， 实验

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), which is an RNA binding protein involved in the transport and translation regulation of specific messenger RNA (mRNA) targets. We have determined that FMRP has nuclease activity, being able to process precursor microRNAs (pre-miRNAs), potentially being involved in the mature miRNA biogenesis. The parental 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. Under this aim we will determine: (i) if the FMRP nuclease activity resides in one of his KH domains and identify the active site residues responsible for this activity by point mutations; (ii) if FMRP S500D has higher nuclease activity as compared with unphosphorylated FMRP due different dimerization properties and (iii) if the 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. Under this specific aim we will: (i) determine if FMRP and FMRP S500D cleave pre-miRNAs into mature miRNAs; (ii) determine if they have additional substrates such as RNA perfect duplex, RNA single strand, RNA G quadruplex, DNA-RNA hybrid duplex, DNA duplex; (iii) characterize the kinetics of their nuclease activity. Similar experiments will pe performed to test the nuclease activity of the FMRP paralogs FXR1P and FXR2P. 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. Under this aim we will investigate if FMRP and FMRP S500D, as well as FXR1P and FXR2P interact with the SARS-CoV-2 3’-UTR genome, cleaving conserved stem- loops either to potentially yield viral miRNAs or functioning in the antiviral host response. Fluorescence spectroscopy will be used in all three specific aims of the parent R15 proposal, with the requested Fluorolog-QM-75-11-C spectrofluorometer and accessories being essential for the success of the proposed experiments.