"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."
RNA is the genetic material that programs cells to make proteins from DNA's blueprint and specifies which proteins should be made. There are many types of RNA in the cells of mammals, such as transfer RNA, ribosomal RNA, and messenger RNA-each with a specific purpose in making and manipulating proteins.
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 Ûlo 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 (mRNA). 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.
The research was supported by grants from the National Institutes of Health, the Swedish Science Foundation, and the European Community. Study coauthors are Jennifer Zielinski, Tiina Peritz, Jeanine Jochems, Theresa Kannanayakal, and Kevin Miyashiro, from Penn, and Kalle Kilk, Emilia Eiriksdóttir, and Ûlo Langel from Stockholm University, Sweden. This release can be found at http://www.
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