A Real Time Look at Interactions Between RNA and
Intracellular Observation of RNA Metabolism Will Help Identify
(Philadelphia, PA) - For the first time, researchers can
now peer inside intact cells to not only identify RNA-binding proteins,
but also observe--in real-time--the intricate activities of these special
molecules that make them key players in managing some of the cell's most
basic functions. Researchers at the University of Pennsylvania
School of Medicine who developed the new technology 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 last week in the 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.”
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
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.
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