Requirement of the Protein B23 for Nucleolar Disassembly Induced by the FRGY2a Family Proteins

• Published in: The Journal of biological chemistry Vol. 281; no. 12; pp. 8153 - 8160
• Main Authors: Gonda, Koichi, Wudel, Justin, Nelson, Dominic, Katoku-Kikyo, Nobuko, Reed, Peter, Tamada, Hiroshi, Kikyo, Nobuaki
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.03.2006; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Cell Line, Tumor; Cell Nucleolus - metabolism; Cell Nucleus - metabolism; Cloning, Molecular; DNA, Complementary - metabolism; HeLa Cells; Histidine - chemistry; Humans; Immunoprecipitation; Mass Spectrometry; Microscopy, Electron; Microscopy, Fluorescence; Nuclear Proteins - metabolism; Nuclear Proteins - physiology; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering - metabolism; RNA-Binding Proteins - metabolism; RNA-Binding Proteins - physiology; Transcription Factors - metabolism; Transcription Factors - physiology; Transfection; Xenopus; Xenopus Proteins - metabolism; Xenopus Proteins - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M512890200

In Xenopus somatic cell nuclear cloning, the nucleoli of donor nuclei rapidly and almost completely disappear in egg cytoplasm. We previously showed that the germ cell-specific proteins FRGY2a and FRGY2b were responsible for this unusually drastic nucleolar disassembly. The nucleolar disassembly occurs without inhibition of pre-rRNA transcription, a well known trigger for nucleolar segregation, and the mechanism for the nucleolar disassembly by FRGY2a and FRGY2b remains largely unknown. In this study, we searched for FRGY2a-interacting proteins and investigated the functional consequences of their interactions through a series of experiments. We showed that during the nucleolar disassembly, FRGY2a localized to the nucleoli of isolated nuclei and was capable of disassembling purified nucleoli, suggesting a direct interaction between FRGY2a and nucleolar components. Using a His tag pulldown approach, we identified the abundant and multifunctional nucleolar protein B23 as a potential target of FRGY2a and its related human protein YB1. A specific interaction between FRGY2a/YB1 and B23 was confirmed by co-immunoprecipitation. Finally, B23 knockdown using short interfering RNA and a subsequent add-back experiment confirmed that B23 was necessary for nucleolar disassembly by YB1. We propose that FRGY2a and YB1 disassemble nucleoli by sequestering B23, which is associated with pre-ribosomes and other structurally important nucleolar components.