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Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding
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| Title: | Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding |
| Authors: | Kitamura, Akira Browse this author →KAKEN DB | | Nakayama, Yusaku Browse this author | | Kinjo, Masataka Browse this author →KAKEN DB |
| Keywords: | Nuclear localization signal | | Nucleolus | | Green fluorescent protein | | Fluorescence recovery after photobleaching | | Fluorescence correlation spectroscopy |
| Issue Date: | 31-Jul-2015 |
| Publisher: | Elsevier |
| Journal Title: | Biochemical and Biophysical Research Communications |
| Volume: | 463 |
| Issue: | 3 |
| Start Page: | 401 |
| End Page: | 406 |
| Publisher DOI: | 10.1016/j.bbrc.2015.05.084 |
| PMID: | 26032495 |
| Abstract: | 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 http://creativecommons.org/licenses/by-nc-nd/4.0/ | | https://creativecommons.org/licenses/by-nc-nd/4.0/ |
| Type: | article (author version) |
| URI: | http://hdl.handle.net/2115/62594 |
| Appears in Collections: | 生命科学院・先端生命科学研究院 (Graduate School of Life Science / Faculty of Advanced Life Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
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Submitter: 北村 朗
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