Mysterious Molecules Begin to Yield Their Secrets

By Jason Socrates Bardi

A team of investigators at The Scripps Research Institute and the Genomics Institute of the Novartis Research Foundation (GNF) have discovered a way to screen hundreds and potentially thousands of "noncoding" RNA molecules to discover their functions within cells.

Unlike traditional RNA, which is copied from DNA to code for a protein, these noncoding RNA molecules are never translated into proteins. But noncoding does not mean uninteresting. There are tens of thousands of noncoding RNA molecules inside human cells, and even if only one percent is functional, that's still hundreds of molecules that may be participating in the control of cellular functions.

In this week's issue of the journal Science, the Scripps Research and GNF team reports on an experimental strategy for searching for these undiscovered functions. As a proof of principal, the scientists screened a library of noncoding RNAs, selected one that seemed to play a cellular role, and performed further experiments to prove that it does.

"We have only just hit the tip of the iceberg," says Professor Peter G. Schultz, who holds the Scripps Family Chair and is a professor in the department of Chemistry and The Skaggs Institute of Chemical Biology at Scripps Research. "There's a whole world of this noncoding RNA."

A Stranger in the Protein Encoding Land

Noncoding RNA has long been regarded as something of a stranger in the protein encoding land of RNA. It falls outside the purview of traditional molecular biologists, who once adhered to a concept called the "central dogma," which held that DNA genes are transcribed into RNA transcripts that are then translated into proteins.

The fact that there is non-coding RNA within cells has been known for several years, but it has only been recently that scientists have begun to appreciate noncoding RNA. Many reports have appeared in the scientific literature describing this RNA that does not encode proteins.

One of the reasons for the turnaround is that scientists have begun to recognize just how abundant noncoding RNA is. In fact, two reports in the same issue of Science this week describe the work of an international consortium of scientists that includes Scripps Florida Professor Claes Wahlestedt showing that there are far more noncoding RNAs than most scientists would have imagined even a few months ago.

The consortium sequenced some 43,553 RNA transcripts isolated from a variety of mammalian cells and tissue. Surprisingly more than half of these RNA transcripts are noncoding. One of the stunning conclusions of these reports is that the amount of noncoding RNA that is being expressed in cells is vast. Wahlestedt and his consortium colleagues found tens of thousands of noncoding RNAs in mammalian cells—a number even larger than the number of protein-encoding genes being expressed. For details on these results, see "Mammalian Transcriptome Mapped, and It Makes Antisense."

So if our DNA is expressing a large number of genes and an even larger number of RNA transcripts that do not code for genes, the question is: why would the cell expend so much energy making RNA if that RNA doesn't do anything? Perhaps some noncoding RNAs play cellular roles. If so, then, what are the noncoding RNAs doing in the cell?

"That's the million-dollar question," says Neurobiology Professor John Hogenesch, who is the director of Genome Technologies at Scripps Florida. "Until now we have not had a generalized way of answering it."

Scientists have speculated as to what these noncoding RNAs are doing based on computational approaches and analyses of the sequences, but nobody has come up with a way of experimentally determining whether the noncoding RNAs have a cellular function.

Proof of a Cellular Function

To apply a high-throughput approach to the problem of detecting the function of certain noncoding RNAs, the Scripps Research and GNF teams began by collecting a library of 512 evolutionarily conserved putative noncoding RNAs. Then, the scientists established screens using a technique called RNA interference to "knock down"—or silence—the noncoding RNA within cells.

The cell-based screens were designed then to look for changes, such as the increase or decrease of activity related to a certain cellular protein. In theory, if this change occurs as a sole result of altering the level of a noncoding RNA, then that noncoding RNA stands a good chance of being involved.

Out of the 512 target noncoding RNAs, the screens returned eight "hits"—eight noncoding RNAs that appeared to have function. Six appeared to affect cell proliferation, one influenced the hedgehog (Hh) signal transduction pathway, and the final one was a strong modifier of nuclear factor of activated T-cells (NFAT) signaling. The team decided to examine in detail this last noncoding RNA. Suspecting that it might interact with proteins, they used a technique to trap the proteins with which the noncoding RNA was interacting. There were ten, a few of which were of the class known as "importins," involved in the transport of materials from the cytoplasm to the cell nucleus.

Among these was the protein "nuclear factor of activated T cells" (NFAT). The scientists found that when they blocked the noncoding RNA, the activity of NFAT increased dramatically. For this reason, they dubbed the noncoding RNA the noncoding repressor of NFAT "NRON."

Demonstrating the function of one noncoding RNA like NRON is only the beginning, says Scripps Research Associate Aaron Willingham, Ph.D., who was the first author on the paper. "You could apply this methodology to look for functions of many other noncoding RNAs."

The article, "A Strategy for Probing the Function of Noncoding RNAs Finds a Repressor of NFAT" is authored by A. T. Willingham, A. P. Orth, S. Batalov, E. C. Peters, B. G. Wen, P. Aza-Blanc, J. B. Hogenesch, and P. G. Schultz and appears in the September 2, 2005 issue of the journal Science. See: www.sciencemag.org.

This work was supported by the Novartis Research Foundation and an NIH Kirschstein National Research Service Award.

Send comments to: jasonb@scripps.edu