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: 10793981
• 负责人: MIHAELA R MIHAILESCU
• 金额: $ 10万
• 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 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10793981
• 关键词: 2019-nCoV; Active Sites; Affect; Biochemical; Biogenesis; Biological Process; COVID-19 pandemic; DNA; Dimerization; FMR1; FXR1 gene; FXR2 gene; Female; Fluorescence Spectroscopy; Fragile X Syndrome; G-Quartets; Genome; Hybrids; Immune response; Inherited; Investigation; KH Domain; Kinetics; Messenger RNA; MicroRNAs; Names; Parents; Play; Point Mutation; Process; Property; Proteins; Psyche structure; RNA; RNA-Binding Proteins; Role; SARS-CoV-2 genome; Syndrome; System; Tertiary Protein Structure; Testing; Translational Regulation; Untranslated Regions; Viral; Virus; antiviral drug development; experimental study; male; novel; nuclease; paralogous gene; protein function; stem; success

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.

项目摘要 脆弱的X心理迟缓综合征是遗传性障碍的最常见形式，影响〜1 在4000名男性中，有6000名女性中的1个。该综合征是由正常细胞蛋白的丧失引起的 脆弱的X智力低下蛋白（FMRP），该蛋白是一种参与运输和的RNA结合蛋白 特定信使RNA（mRNA）靶标的翻译调节。我们已经确定FMRP具有核酸酶 活性，能够处理前体microRNA（前MIRNA），可能参与成熟 miRNA生物发生。父母提案将表征这种新颖的FMRP功能，具有以下 具体目的： Aim I.识别FMRP域负责其核酸酶活性。在这个目标下，我们将 确定：（i）FMRP核酸酶活性是否存在于他的KH域之一并确定活性位点残差 负责该活动的点突变； （ii）如果FMRP S500D具有较高的核酸酶活性 未磷酸化的FMRP应有不同的二聚化特性，并且（iii）如果旁系同源物fxr1p和fxr2p， 与FMRP共享KH0，KH1和KH2结构域也具有核酸酶活性。 目标II。 FMRP核酸酶活性的生化表征。在这个具体目标下，我们将：（i） 确定FMRP和FMRP S500D是否将Pre MiRNA清除到成熟的miRNA中； （ii）确定他们是否有 其他底物，例如RNA Perfect Duplex，RNA单链，RNA G四链体，DNA-RNA杂交 双链体，DNA双链体； （iii）表征其核酸酶活性的动力学。类似的实验将进行PE进行 测试FMRP旁系同源物FXR1P和FXR2P的核酸酶活性。 目标三。 FMRP与SARS-COV-2 3'-UTR基因组的相互作用的研究和潜力 其核酸酶活性在此病毒系统中扮演的角色。在此目标下，我们将调查FMRP和FMRP是否 S500D以及FXR1P和FXR2P与SARS-COV-2 3'-UTR基因组相互作用，分裂构成茎 - 循环可以产生潜在的病毒miRNA或在抗病毒宿主反应中起作用。 荧光光谱将用于母体R15提案的所有三个特定目的，并请求 Fluorolog-QM-75-11-C Spectrofluorometer和配件对于所提出的成功至关重要 实验。

项目成果

Effect of the SARS-CoV-2 Delta-associated G15U mutation on the s2m element dimerization and its interactions with miR-1307-3p

SARS-CoV-2 Delta 相关 G15U 突变对 s2m 元件二聚化的影响及其与 miR-1307-3p 的相互作用

• DOI: 10.1261/rna.079627.123
• 发表时间: 2023
• 期刊: RNA
• 影响因子: 4.5
• 作者: Cunningham, Caylee L.;Frye, Caleb J.;Makowski, Joseph A.;Kensinger, Adam H.;Shine, Morgan;Milback, Ella J.;Lackey, Patrick E.;Evanseck, Jeffrey D.;Mihailescu, Mihaela-Rita
• 通讯作者: Mihailescu, Mihaela-Rita