Reversible disassembly of the nucleolus by FRGY proteins

描述（申请人提供）：核仁是一个动态的细胞器。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，核仁蛋白在核仁和核质之间不断快速穿梭。然而，这些核仁动力学背后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。研究人员最近在体外和体内发现爪蟾生殖细胞蛋白FRGY2a和FRGY2b可以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。生理上，FRGY2a/b和人类同源物YB1对有丝分裂核仁拆卸、活体细胞核仁和核质之间的蛋白质穿梭以及癌症化疗药物诱导的核仁拆卸至关重要。FRGY2a/b和YBI对核仁分解的进一步研究提出了三个具体目标。（SA1）确定FRGY2a/b和YBI在核仁拆卸中的生理作用。在化学核仁拆卸、有丝分裂核仁拆卸和核仁蛋白穿梭的背景下，YB1复合物的核仁拆卸活性和磷酸化将在人类体细胞中进行研究。将通过微量注射FRGY2a/b显性阴性突变体来研究早期非洲爪蟾胚胎中拆卸核仁的维持。（SA2）了解YB1破坏核仁的分子机制。利用免疫荧光显微镜和电子显微镜研究YB1的核仁拆卸过程和亚核仁定位。在核仁分解过程中与YB1相互作用的蛋白将通过亲和纯化分离。这些蛋白的功能将通过鉴定亚核定位、上调（通过转染）和下调（通过短干扰RNA和反义）来研究。YB1磷酸化对与相互作用蛋白结合的影响也将被研究。（SA3）鉴定和表征卵母细胞提取物中FRGY2a/b的抑制剂。在排卵之前，爪蟾卵被称为卵母细胞，它们与卵不同，有多个核仁。卵母细胞含有FRGY2a/b及其抑制剂。采用免疫亲和纯化、his标签下拉和常规柱纯化分离抑制剂。我们将通过分析其表达模式、上调和下调来研究该抑制剂的作用，重点关注间期细胞核仁的维持和末期核仁的重组。这些项目很重要，因为最近的研究表明，核核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。该研究结果将有助于了解核仁动力学，使我们能够调节其多种功能，以造福于医学。

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.