Gonda Koichi, Wudel Justin, Nelson Dominic, Katoku-Kikyo Nobuko, Reed Peter, Tamada Hiroshi, Kikyo Nobuaki
Stem Cell Institute, Division of Hematology, Oncology and Transplantation, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, USA.
J Biol Chem. 2006 Mar 24;281(12):8153-60. doi: 10.1074/jbc.M512890200. Epub 2006 Jan 16.
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.
在非洲爪蟾体细胞克隆中，供体细胞核的核仁在卵细胞质中迅速且几乎完全消失。我们之前表明，生殖细胞特异性蛋白FRGY2a和FRGY2b是这种异常剧烈的核仁解体的原因。核仁解体在不抑制前体核糖体RNA转录（一种众所周知的核仁分离触发因素）的情况下发生，并且FRGY2a和FRGY2b导致核仁解体的机制在很大程度上仍然未知。在本研究中，我们寻找与FRGY2a相互作用的蛋白，并通过一系列实验研究了它们相互作用的功能后果。我们表明，在核仁解体过程中，FRGY2a定位于分离细胞核的核仁，并能够拆解纯化的核仁，这表明FRGY2a与核仁成分之间存在直接相互作用。使用His标签下拉方法，我们鉴定出丰富且多功能的核仁蛋白B23是FRGY2a及其相关人类蛋白YB1的潜在靶点。通过免疫共沉淀证实了FRGY2a/YB1与B23之间存在特异性相互作用。最后，使用小干扰RNA敲低B23以及随后的回补实验证实，B23是YB1介导核仁解体所必需的。我们提出，FRGY2a和YB1通过隔离与前核糖体及其他结构上重要的核仁成分相关联的B23来拆解核仁。
