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Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding


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タイトル: Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding
著者: Kitamura, Akira 著作を一覧する
Nakayama, Yusaku 著作を一覧する
Kinjo, Masataka 著作を一覧する
キーワード: Nuclear localization signal
Green fluorescent protein
Fluorescence recovery after photobleaching
Fluorescence correlation spectroscopy
発行日: 2015年 7月31日
出版者: Elsevier
誌名: Biochemical and Biophysical Research Communications
巻: 463
号: 3
開始ページ: 401
終了ページ: 406
出版社 DOI: 10.1016/j.bbrc.2015.05.084
抄録: Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS(SV40)) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS(SV40) in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS(SV40) formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS(SV40) likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS(SV40) can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells.
Rights: ©2016. This manuscript version is made available under the CC-BY-NC-ND 4.0 license
資料タイプ: article (author version)
出現コレクション:雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

提供者: 北村 朗


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