SAMIE JAFFREY AND JEREMY PAIGE, CORNELL UNIVERSITY
Even with the amazing array of tools available to biologists studying cellular functions, watching these processes unfold inside a living cell remains a challenge. Green fluorescent protein (GFP) revolutionized imaging and the spatial understanding of how proteins interact within living cells, but a lot of information is also embedded within the production, processing, and manifold interactions of RNA.
Researchers are using RNA tracking systems to follow the paths of noncoding RNAs, the transcription of mRNA, splicing patterns, and the trafficking of RNA in development and in RNA-based disease processes. But as with all imaging techniques, you have to consider whether the labeling method will interfere with the process that you’d like to study, says Bruce Armitage, a chemist at Carnegie Mellon University in Pittsburgh, Pennsylvania. “While GFP is extremely useful, there is always a concern when you put a large tag onto your protein that maybe it behaves differently than it does when that tag is not there. Since you can’t see it if the tag’s not there, you don’t always know if the tag is misinforming you,” he says. “The same is true with almost any RNA technology.”
Many RNA tracking techniques are still in development. To find what works best for your experimental system, “you have to go into this expecting to try a few different technologies,” Armitage says. That will require a bit of detective work. Some reagents are available for order from companies, but you may also need to contact principal investigators on the methods papers, either to get reagents or to learn more about how to carry out the experiment.
“Maybe five to ten years from now we’ll have a robust enough technology and enough experimental testing that there will be a nice clearinghouse for information and [researchers will] get their ideal probe,” Armitage says. Until then, to get you started, here’s a guide to the main techniques for tracking RNA within living cells.
TAGGING RNA WITH GFP
The original technique for tracking RNA inside living cells, developed about 15 years ago, uses GFP as the fluorescent reporter molecule. Previously, researchers had used the coat protein from the MS2 bacteriophage, which binds to a defined 19-nucleotide sequence of RNA, as a way to capture RNA and RNA-binding proteins. By fusing GFP to this MS2 protein, Robert Singer and his colleagues at Albert Einstein College of Medicine in Bronx, New York, first demonstrated that they could follow RNA as it migrated within cells (Molecular Cell, 2:437-45, 1998).
The first step is adding copies of MS2’s RNA-binding sequence, which forms a characteristic stem loop structure, to the noncoding region of the gene of interest. In addition, the cells need to be transfected with a plasmid so that they can express the MS2-GFP fusion protein. There’s also an inherent problem to surmount—the background fluorescence of the fusion protein before it binds to the target RNA. Singer and his colleagues realized that they’d need to insert many copies—24, and sometimes even 48—of the MS2 stem loop within the RNA of interest to overcome this “haze of GFP” and get sufficient signal to track a single molecule moving within the cell in real time.
Researchers have used the technique in many different cell types, including yeast, Drosophila, and various mammalian cells. Singer and his colleagues have even developed MS2-GFP transgenic mice that track mRNA generated by the ß-actin gene (Nat Methods, 8:165-70, 2011). They are currently developing a second transgenic mouse, and others could use the technique to develop their own mouse models.
Pros & Cons
On the plus side, because the probes are expressed within the cell, you don’t have to worry about delivering large, highly charged reagents across the cell membrane. One potential problem, however, is that the MS2 protein includes a nuclear trafficking signal, prompting questions about whether that signal could modify the localization behavior of the RNA-protein complex. Right now, because there is only one targ