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Techniques for following the movement of single RNAs in living cells

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Abstract The ability to investigate gene expression has evolved from static approaches that analyze a population of cells to dynamic approaches that analyze individual living cells. During the last decade, a number of different fluorescent methods have been developed for monitoring the dynamics of single RNAs in living cells. Spatial–temporal analyses of single RNAs in living cells have provided novel insight into nuclear transport, RNA localization, and decay. Technical advances with these approaches allow for single molecule detection, providing an unprecedented view of RNA movement. In this article, we discuss the methods for observing single RNAs in living cells, highlighting the advantages and limitations of each method. WIREs RNA 2011 2 601–609 DOI: 10.1002/wrna.83 This article is categorized under: RNA Export and Localization > Nuclear Export/Import RNA Export and Localization > RNA Localization RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms

Visualizing RNA in living cells with the MS2‐GFP system. (a) Localization of a lacZ reporter mRNA containing the ASH1 E3 cis‐acting localization element and six MS2 stem‐loop structures in the yeast Saccharomyces cerevisiae. The small particle in the bud is the localized lacZ reporter mRNA, while unbound MS2‐GFP is concentrated in the mother cell nucleus. (b) Depiction of the MS2‐GFP system. The ‘gray balls’ represent MS2 and the ‘green balls’ represent GFP. As MS2 protein forms dimers, six MS2 stem‐loop structures could contain 12 MS2‐GFP fusion proteins.

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Visualizing RNA in living cells with molecular beacons. (a) Localization of oskar mRNA (red) and actin (green) in a wild‐type Drosophila oocyte. (b) Schematic representation of a molecular beacon. When a molecular beacon folds into a stem‐loop conformation, the quencher, gray ball, is brought into close proximity of the fluorophore, green ball, preventing fluorescence. Upon hybridization of the molecular beacon to the target RNA, the distance between the quencher and the fluorophore increases, allowing detection of the emitted light. (c) Schematic representation of fluorescence resonance energy transfer (FRET) using molecular beacons. When two molecular beacons simultaneously hybridize to the target RNA, the emitted energy from one fluorophore (green ball) can excite a second fluorophore (red ball), resulting in the emission of light.

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RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms
RNA Export and Localization > Nuclear Export/Import
RNA Export and Localization > RNA Localization

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