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.
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