Researchers at the University of Pennsylvania School of Medicine have announced that they have developed a technology to peer inside intact cells to not only identify RNA-binding proteins, but also observe-in real-time-the intricate activities of these special molecules, see this advance as one of the next logical steps in genomics research.
Senior author James Eberwine, PhD, Professor of Pharmacology at Penn, and colleagues published their research in an issue of Proceedings of the National Academy of Sciences.
"Now we have a workable system to understand all aspects of RNA metabolism in a cell," say Eberwine.
"For the first time, we can study how manipulation of cellular physiology, such as administering a drug, changes RNA-binding protein and RNA interactions. This technology allows us to see that in real time in real cells."
The workhorses of the cell, RNA-binding proteins regulate every aspect of RNA function.
Indeed, RNA is transported from one site to another inside the cell by RNA-binding proteins; RNA is translated into protein with the help of RNA-binding proteins, and RNA-binding proteins degrade used RNA.
"They're really the master regulators of expression in the cell," says Eberwine.
Using whole neurons from rodents, the researchers were able to identify RNA interactions in live cells.
In collaboration with Ulo Langel from Stockholm University, the Penn investigators devised a many-talented molecule that does not get broken down by enzymes once inside a live cell.
One end of the molecule, called a peptide nucleic acid (PNA), has a cell-penetrating peptide called transportan 10 to first get the PNA through the cell membrane.
Once in the cell, the PNA binds to a specific target messenger RNA. There is also a compound on the molecule that can be activated by light and will cross-link the PNA to whatever protein is nearby.
The researchers isolated an array of proteins from the complex of the PNA, the targeted mRNAs, and associated RNA-binding proteins.
The cells are then broken apart and the proteins that interact with the mRNA are identified with a mass spectrometer.
With their system, the researchers are trying to identify RNA-binding proteins that bind RNAs of interest-such as those involved in the targeting, degradation, and translation of RNAs into proteins.
Once identified, the Eberwine team uses another technology they developed to find the other RNA cargos that bind to that RNA-binding protein. These are other RNAs that likely co-regulate RNAs associated with disease.