RRC ID 52779
Author Kitamura A, Nakayama Y, Kinjo M.
Title Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding.
Journal Biochem Biophys Res Commun
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.
Volume 463(3)
Pages 401-6
Published 2015-7-31
DOI 10.1016/j.bbrc.2015.05.084
PII S0006-291X(15)30008-5
PMID 26032495
MeSH Amino Acid Sequence Animals Cell Line, Tumor Cell Nucleus / metabolism* Cell Nucleus / ultrastructure Cytoplasm / metabolism Cytoplasm / ultrastructure Fluorescence Recovery After Photobleaching Green Fluorescent Proteins / analysis* Green Fluorescent Proteins / metabolism Mice Molecular Sequence Data Nuclear Localization Signals / analysis* Nuclear Localization Signals / metabolism RNA / metabolism* Spectrometry, Fluorescence
IF 2.705
Times Cited 7
DNA material mCherry-fibrillarin (RDB15739) GFP-NLS SV40 (RDB15740) GFP-NLS PARP (RDB15741).