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和准确地重组。在间期细胞中，核仁蛋白持续快速地在核仁和核质之间穿梭。然而，这些核仁动力学背后的分子机制还不清楚。这项研究的长期目标是确定核仁组织组装和拆解的分子基础。研究人员最近发现，非洲爪哇生殖细胞蛋白FRGY2a和FRGY2b在体外和体内都可以可逆地分解体细胞核仁。它们是第一批具有这种能力的蛋白质。从生理上讲，FRGY2a/b和人类同系物YB1是有丝分裂核仁解体、蛋白质在活体细胞核仁和核质之间穿梭以及癌症化疗药物诱导的核仁解体所必需的。为进一步研究FRGY2a/b和YBI对核仁的拆解，提出了三个具体的目标。(SA1)建立FRGY2a/b和YBI对核仁分解的生理作用。在化学核仁分解、有丝分裂核仁分解和核仁蛋白质穿梭的背景下，将使用人体细胞研究YB1复合体的核仁分解活性和磷酸化。通过显微注射显性负向突变体FRGY2a/b(SA2)来研究早期非洲爪哇胚胎中分解核仁的维持。了解YB1分解核仁的分子机制。用免疫荧光显微镜和电子显微镜研究YB1的核仁解体过程和亚核仁定位。在核仁解体过程中与YB1相互作用的蛋白质将通过亲和纯化来分离。这些蛋白质的功能将通过鉴定这些蛋白质在细胞内的亚核定位、上调和下调(通过短干扰RNA和反义)来研究。YB1的磷酸化对与相互作用蛋白结合的影响也将被研究。(SA3)鉴定卵母细胞提取液中FRGY2a/b的抑制物S。非洲爪哇的卵在排卵之前被称为卵母细胞，与卵不同的是，它们有多个核仁。卵母细胞含有FRGY2a/b及其抑制物(S)。将采用免疫亲和纯化、His-tag下拉和常规柱纯化的方法来分离该抑制物。通过对其表达模式、上调和下调的分析，将研究该抑制物在间期细胞中核仁的维持和末期核仁的重组中的作用。这些项目之所以重要，是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。这项拟议的研究结果将大大有助于理解核仁的动力学，使我们能够调节其不同的功能，从而达到医疗目的。

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.