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In Situ Hybridization for the Precise Localization of Transcripts in Plants

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Visualizing nuclear-localized RNA using transient expression system in plants.

Natsuko Kinoshita¹, Catalina Arenas-Huertero¹, Nam-Hai Chua¹

¹Lab of Plant Molecular Biology, The Rockefeller University, New York, NY, USA.

Genes to Cells : Devoted to Molecular & Cellular Mechanisms

|December 23, 2017

Summary

Researchers developed a new method for visualizing nuclear RNA by modifying an existing cytosolic RNA tool. This technique uses MS2 sequences and modified MS2 coat proteins to track RNA within the cell nucleus.

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Area of Science:

Molecular Biology
Cell Biology
Genetics

Background:

Existing tools for visualizing RNA are primarily limited to the cytosol.
Accurate visualization of nuclear RNA is crucial for understanding gene regulation and cellular processes.
Previous methods for nuclear RNA tracking faced challenges with background signal and efficiency.

Purpose of the Study:

To develop a novel and improved method for visualizing RNA localized within the cell nucleus.
To overcome limitations of existing RNA visualization techniques, particularly concerning nuclear background noise.
To enhance the efficiency and reliability of RNA tracking in transient expression systems.

Main Methods:

Modification of a cytosolic RNA visualization tool using bacteriophage-derived MS2 sequences.
Fusion of the RNA of interest to MS2 sequences and Green Fluorescent Protein (GFP) fused to MS2 coat protein (MSCP).
Strategic use of a nuclear export sequence (NES) to control MSCP-GFP probe localization and reduce nuclear background, and addition of mCherry as a visual marker.

Main Results:

Successful development of a method for visualizing nuclear-localized RNA.
Demonstrated reduction of nuclear background signals through the use of an NES.
Enhanced efficiency of the RNA visualization probe in transient systems due to the addition of mCherry.

Conclusions:

The developed method provides a robust approach for visualizing nuclear RNA.
The strategic use of NES and mCherry improves the specificity and efficiency of nuclear RNA tracking.
This advancement offers new possibilities for studying nuclear RNA dynamics and function.