Reversible disassembly of the nucleolus by FRGY proteins

描述（申请人提供）：核仁是一个动态细胞器。在高等真核生物的有丝分裂过程中，其高度组织化的结构被完全分解并精确地重新组装。在间期细胞中，核仁蛋白质不断快速地穿梭于核仁和核质之间。然而，这些核仁动力学的分子机制是不明确的。本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FRGY 2a和FRGY 2b在体外和体内都能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。在生理学上，FRGY 2 a/B和人类同源物YB 1对于有丝分裂核仁解体、活体细胞中核仁和核质之间的蛋白质穿梭以及由癌症化疗药物诱导的核仁解体是必需的。提出了进一步研究FRGY 2a/B和YBI的核仁解体的三个具体目标。(SA1)通过FRGY 2a/B和YBI建立核仁解体的生理作用。将使用人类体细胞研究化学核仁解体、有丝分裂核仁解体和核仁蛋白穿梭背景下的核仁解体活性和YB 1复合物的磷酸化。将通过显微注射FRGY 2a/B的显性失活突变体来研究早期爪蟾胚胎中解体的核仁的维持。(SA2)了解YB 1引起核仁解体的分子机制。用免疫荧光显微镜和电子显微镜研究YB 1的核仁解体过程和亚核仁定位。在核仁解体过程中与YB 1相互作用的蛋白质将通过亲和纯化分离。这些蛋白质的功能将通过鉴定细胞内蛋白质的亚核定位、上调（通过转染）和下调（通过短干扰RNA和反义）来研究。还将研究YB 1磷酸化对与相互作用蛋白结合的影响。(SA3)鉴定和表征卵母细胞提取物中FRGY 2a/B的抑制剂。爪蟾卵在排卵前被称为卵母细胞，它们有多个核仁，不像卵。卵母细胞含有FRGY 2 a/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。通过分析其表达模式、上调和下调，将研究该抑制剂的作用，重点是维持间期细胞中的核仁和末期细胞中的核仁重新组装。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，而且还包括细胞生物学中更广泛的领域，如细胞周期控制，癌细胞增殖和端粒酶调节。拟议研究的结果将大大有助于理解核仁动力学，使我们能够调节其多种功能，以获得医疗效益。

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.