Reversible disassembly of the nucleolus by FRGY proteins

FRGY 蛋白对核仁的可逆分解

基本信息

批准号：
7485281
负责人：
Nobuaki Kikyo
金额：
$ 9.51万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-30 至 2009-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7485281
关键词：
Affinity Chromatography Antisense RNA Area Binding Biological Assay Cell Cycle Regulation Cell Nucleolus Cell Proliferation Cells Cellular biology Chemicals Chemotherapy-Oncologic Procedure Cloning Complex Dominant-Negative Mutation Down-Regulation Drug usage Electron Microscopy Embryo Eukaryota Eukaryotic Cell Future Genetic Transcription Germ Cells Goals Homologous Gene Human Immunofluorescence Microscopy In Vitro Interphase Interphase Cell Knowledge Laboratories Life Localized Maintenance Medical Microinjections Mitosis Mitotic Molecular Nucleolar Proteins Nucleoplasm Oocytes Organelles Outcome Ovulation Pattern Pharmaceutical Preparations Phosphorylation Physiological Play Positioning Attribute Process Proteins Range Reagent Regulation Research Research Personnel Ribosomal DNA Ribosomal RNA Ribosomes Role Somatic Cell Structure Telomerase Transcription Process Transfection Xenopus Xenopus oocyte base cancer cell casein kinase II egg in vitro Model in vivo inhibitor/antagonist novel nuclear transfer protein function protein protein interaction telophase

项目摘要

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.

核仁是一个动态的细胞器。其高度组织化的结构完全可拆卸且精确在高等真核生物有丝分裂期间重新组装。在间期细胞中，核仁蛋白不断且快速地产生核仁和核质之间穿梭。然而，这些核仁背后的分子机制动力学的定义不明确。这项研究的长期目标是确定组装和拆卸的分子基础核仁组织。研究人员最近发现非洲爪蟾生殖细胞蛋白 FRGY2a 和 FRGY2b 可以在体外和体内可逆地分解体细胞核仁。它们是第一个具有这种能力的蛋白质。生理学上，FRGY2a/b 和人类同源物 YB1 对于有丝分裂核仁解体、蛋白质活体细胞中核仁和核质之间的穿梭以及癌症引起的核仁解体化疗药物。提出了三个具体目标来进一步研究 FRGY2a/b 和 YBI 的核仁分解。 (SA1)确定FRGY2a/b和YBI对核仁分解的生理作用。核仁拆卸活性和化学核仁拆卸、有丝分裂核仁拆卸背景下 YB1 复合物的磷酸化将使用人类体细胞研究核仁蛋白穿梭。早期拆卸核仁的维持将通过显微注射 FRGY2a/b 显性失活突变体来研究非洲爪蟾胚胎。 (SA2) 了解 YB1 核仁分解的分子机制。核仁拆卸过程和亚核仁定位 YB1 将通过免疫荧光显微镜和电子显微镜进行研究。与 YB1 相互作用的蛋白质在核仁解体过程中，将通过亲和纯化来分离。这些蛋白质的功能将通过以下方式进行研究鉴定亚核定位、上调（通过转染）和下调（通过短干扰 RNA 和细胞内蛋白质的反义）。 YB1 磷酸化对相互作用蛋白结合的影响将是也研究过。 (SA3) 鉴定并表征卵母细胞提取物中 FRGY2a/b 的抑制剂。爪蟾卵被称为卵母细胞直到排卵，与鸡蛋不同，它们有多个核仁。卵母细胞含有 FRGY2a/b 及其抑制剂。将采用免疫亲和纯化、His 标签下拉和常规柱纯化来分离抑制剂。抑制剂的作用重点是维持间期细胞的核仁和重组将通过分析其表达模式、上调和下调来研究末期核仁。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，还包括涵盖细胞生物学更广泛的领域，例如细胞周期控制、癌细胞增殖和端粒酶规定。拟议研究的结果将极大地有助于理解核仁动态，使我们能够调节其多种功能以获得医疗利益。