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Visualizing nuclear‐localized RNA using transient expression system in plants
Author(s) -
Kinoshita Natsuko,
ArenasHuertero Catalina,
Chua NamHai
Publication year - 2018
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/gtc.12549
Subject(s) - rna , cytosol , nucleus , biology , nuclear localization sequence , green fluorescent protein , microbiology and biotechnology , messenger rna , nuclear transport , cell nucleus , biochemistry , gene , enzyme
By modifying the existing cytosolic RNA visualization tool pioneered by Schönberger, Hammes, and Dresselhaus (2012), we developed a method to visualize nuclear‐localized RNA . Our method uses (i) an RNA component that consists of an RNA of interest that is fused to a bacteriophage‐derived MS 2 sequence; and (ii) GFP fused to MS 2 coat protein ( MSCP ), which binds specifically to MS 2 as is also the case in the method for cytosolic RNA visualization. The nuclear localization sequence ( NLS ) at the C‐terminal of MSCP ‐ GFP tethers the probe to the nucleus. To reduce background signals in the nucleus, we replaced the NLS with a nuclear export sequence ( NES ) that anchors the MSCP ‐ GFP probe in the cytosol. Our nuclear RNA visualization method differs from previous methods in two aspects: (i) We used an NES to reduce nuclear background signal so that the MSCP ‐ GFP probe localizes in the cytosol by default; (ii) We added mC herry as a visual marker in the RNA component to increase its efficient usage in a transient system.