title: Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding.
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.
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kitamura
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Nakayama
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan. Electronic address: kinjo@sci.hokudai.ac.jp.
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000032:analysis* / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D019913:Nuclear Localization Signals; D049452:Green Fluorescent Proteins; D012313:RNA
keywords: Fluorescence correlation spectroscopy; Fluorescence recovery after photobleaching; Green fluorescent protein; Nuclear localization signal; Nucleolus