Imaging of Endogenous RNA Using Genetically Encoded Probes
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- Abstract
- Table of Contents
- Materials
- Figures
- Literature Cited
Abstract
Imaging of RNAs in single cells revealed their localized transcription and specific function. Such information cannot be obtained from bulk measurements. This unit contains a protocol of an imaging method capable of visualizing endogenous RNAs bound to genetically encoded fluorescent probes in single living cells. The protocol includes methods of design and construction of the probes, their characterization, and imaging a target RNA in living cells. The methods for RNA imaging are generally applicable to many kinds of RNAs and may allow for elucidating novel functions of localized RNAs and understanding their dynamics in living cells. Curr. Protoc. Chem. Biol. 3:27?37 © 2011 by John Wiley & Sons, Inc.
Keywords: RNA; imaging; GFP; fluorescence; molecular beacon
Table of Contents
- Introduction
- Strategic Planning
- Basic Protocol 1: Characterization of RNA Probes
- Basic Protocol 2: Imaging Endogenous RNA Using Genetically Engineered Fluorescent Probes
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
Materials
Basic Protocol 1: Characterization of RNA Probes
Materials
Basic Protocol 2: Imaging Endogenous RNA Using Genetically Engineered Fluorescent Probes
Materials
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Figures
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Figure 1. (A ) Structure of the human PUM‐HD complexed with RNA. The helical repeats are shown alternately blue and yellow, which are labeled as repeat 1(R1) to repeat 8(R8). Each repeat recognizes a specific base of RNA. (B ) Basic principle of the RNA probes. Two RNA‐binding domains of PUM are engineered to recognize specific sequences on a target mRNA (mPUM1 and mPUM2). In the presence of the target mRNA, mPUM1 and mPUM2 bind to their target sequences, bringing together the N‐ and C‐terminal fragments of EGFP, resulting in functional reconstitution of the fluorescent protein. (C ) RNA sequences of PUM‐HD for RNA. The amino acids that interact with RNA bases are shown. The amino acids in square frames are necessary for stacking between upper and lower RNA bases. The other amino acids are for hydrogen bonds or van der Waals interactions. View Image -
Figure 2. Flow chart of RNA‐probe characterization. View Image -
Figure 3. Constructs of the plasmids. FLAG, FLAG epitope; MTS, matrix‐targeting signal derived from subunit VIII of cytochrome C oxidase. The cDNA is inserted into an expression vector. View Image -
Figure 4. Fluorescence images of HeLa cells expressing GN‐mPUM1 and mPUM2‐GC stained with MitoTracker and DAPI: (A ) Localization of mitochondria, (B ) reconstituted EGFP, and (C ) mtDNA. Panels D and E show their merged images. Bar, 5 µm. The insets are enlarged images of the boxed region of (A) (bar, 1.2 µm). White arrows indicate colocalization of mtDNA and ND6 mRNA. View Image
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Literature Cited
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