Reversible disassembly of the nucleolus by FRGY proteins

FRGY 蛋白对核仁的可逆分解

• 批准号: 7118085
• 负责人: Nobuaki Kikyo
• 金额: $ 25.81万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118085
• 关键词: Xenopus oocyte; antisense nucleic acid; cell cycle; electron microscopy; enzyme inhibitors; fluorescence microscopy; immunoaffinity chromatography; immunofluorescence technique; intracellular transport; microinjections; molecular assembly /self assembly; nucleolus; phosphorylation; protein kinase; protein protein interaction; protein structure function; protein transport; tissue /cell culture

DESCRIPTION (provided by applicant): The nucleolus is a dynamic organelle. Its highly organized structure is completely disassembled and accurately reassembled during mitosis in higher eukaryotes. In interphase cells, nucleolar proteins are constantly and rapidly shuttling between the nucleolus and nucleoplasm. However, molecular mechanisms underlying these nucleolar dynamics are ill defined. The long-term goal of this research is to identify molecular basis of assembly and disassembly of the nucleolar organization. The investigators have recently found that Xenopus germ cell proteins FRGY2a and FRGY2b can reversibly disassemble somatic nucleoli in vitro and in vivo. They are the first proteins with this capability. Physiologically, FRGY2a/b and the human homologue YB1 are essential for mitotic nucleolar disassembly, protein shuttling between nucleoli and nucleoplasm in living somatic cells and nucleolar disassembly induced by a cancer chemotherapy drug. Three specific aims are proposed to further study nucleolar disassembly by FRGY2a/b and YBI. (SA1) Establish physiological roles of nucleolar disassembly by FRGY2a/b and YBI. Nucleolar disassembly activity and phosphorylation of the YB1 complex in the context of chemical nucleolar disassembly, mitotic nucleolar disassembly and nucleolar protein shuttling will be studied using human somatic cells. Maintenance of disassembled nucleoli in early Xenopus embryos will be studied by microinjection of dominant negative mutants of FRGY2a/b. (SA2) Understand the molecular mechanisms of nucleolar disassembly by YB1. Nucleolar disassembly process and subnucleolar localization of YB1 will be studied with immunofluorescence microscopy and electron microscopy. Proteins interacting with YB1 during nucleolar disassembly will be isolated by affinity purification. Functions of these proteins will be studied through identification of subnuclear localization, up- (by transfection) and down-regulation (by short interfering RNA and antisense) of the proteins within cells. Effects of YB1 phosphorylation on the binding to the interacting proteins will be also studied. (SA3) Identify and characterize inhibitor(s) of FRGY2a/b in oocyte extract. Xenopus eggs are called oocytes until ovulation and they have multiple nucleoli unlike eggs. Oocytes contain FRGY2a/b and its inhibitor(s). Immunoaffinity purification, His-tag pull-down and conventional column purification will be employed to isolate the inhibitor. Roles of the inhibitor with the emphasis on maintenance of nucleoli in interphase cells and reassembly of nucleoli in telophase will be investigated by analysis of its expression pattern, up- and down-regulation. These projects are important because recent studies show that the function of the nucleolus is not limited to ribosome synthesis but encompasses more wide areas in cell biology such as cell cycle control, cancer cell proliferation and telomerase regulation. The outcome of the proposed research will significantly contribute to the understanding of the nucleolar dynamics, enabling us to regulate its diverse functions for medical benefits.

描述（由申请人提供）：核仁是动态细胞器。其高度组织的结构完全拆卸，并在高等真核生物中有丝分裂过程中精确重新组装。在相间细胞中，核仁蛋白在核仁和核质之间不断地迅速穿梭。然而，这些核仁动力学基础的分子机制是错误的定义。这项研究的长期目标是确定核仁组织组装和拆卸的分子基础。研究人员最近发现，爪蟾生殖细胞蛋白frgy2a和frgy2b可以在体外和体内可逆地拆卸体细胞核。它们是具有此能力的第一个蛋白质。在生理上，FRGY2A/B和人类同源YB1对于有丝分裂核仁拆卸至关重要，活体细胞中的核仁和核质之间的蛋白质穿梭以及由癌症化学疗法药物诱导的核仁拆分。提出了三个特定的目的，以进一步研究FRGY2A/B和YBI的核酸拆卸。 （SA1）通过FRGY2A/B和YBI建立核仁拆解的生理作用。在化学核仁拆解，有丝分裂核仁拆解和核仁蛋白穿梭的背景下，YB1复合物的核仁拆解活性和磷酸化将使用人类体细胞进行研究。通过微注射FRGY2A/b的显性阴性突变体的显微注射，将研究脱组合的核仁的维持。 （SA2）了解YB1核酸拆卸的分子机制。将使用免疫荧光显微镜和电子显微镜研究YB1的核仁拆卸过程和YB1的亚核定质定位。在核仁分解过程中与YB1相互作用的蛋白将通过亲和力纯化分离。这些蛋白质的功能将通过鉴定在细胞内蛋白质的蛋白质下（通过转染）和下调（通过简短干扰RNA和反义）来研究。还将研究YB1磷酸化对与相互作用蛋白结合的影响。 （SA3）在卵母细胞提取物中识别和表征FRGY2A/B的抑制剂。爪蟾卵被称为卵母细胞，直到排卵，并且与卵不同。卵母细胞含有FRGY2A/B及其抑制剂。免疫亲和力净化，HIS标签下拉和常规柱净化将用于隔离抑制剂。抑制剂的作用是通过分析其表达模式，向上和下调来研究抑制剂在相间细胞中维持核仁的维持和核仁重新组装的作用。这些项目很重要，因为最近的研究表明，核仁的功能不仅限于核糖体合成，而包括细胞生物学中更广泛的区域，例如细胞周期控制，癌细胞增殖和端粒酶调节。拟议研究的结果将显着有助于理解核仁动态，从而使我们能够调节其多样化的功能以获得医疗益处。