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The RNA behind brain disease

Researchers describe a new technique for cataloging genetic targets in neurological disorders

BY JOSEPH BONNER

Imagine you needed to make a list of every person you had ever called on the phone. It’s a challenge akin to the one faced recently by Robert Darnell’s lab, which studies diseases of the brain.

In order to identify the pathways responsible for disorders such as fragile-X mental retardation, autoimmune neurodegenerative disease and ataxia, they had to create a list of all the RNA segments in a cell that ever bind to a specific protein. Current techniques for identifying such RNA targets are no better than pushing redial; even when they work they tell only a small fraction of the story.

The scientists’ solution was to invent a new technique. Called ultraviolet cross-linking and immunoprecipitation (or CLIP), their new procedure tweaks and combines a pair of standard biochemical techniques to obtain better results. CLIP is the first technique that gives scientists the potential to rapidly identify all the RNAs that bind to a particular RNA-binding protein.

The technique works by exposing cells to ultraviolet light, which causes very strong chemical bonds — “irreversible links” — to develop between RNA and the proteins with which it makes direct contact. After exposing mouse brain tissue to UV light, the scientists used a technique called immunoprecipitation to purify the samples using antibodies.

In a paper published last month in the journal Science, Darnell and colleagues in his Laboratory of Molecular Neuro-Oncology used the CLIP technique on samples taken from patients at The Rockefeller University Hospital to identify 340 RNA segments that bind to a protein called Nova. (By comparison, previously available methods involving work done over the last seven years yielded just three targets.)

Darnell’s lab had previously shown that Nova regulated alternative splicing, the process by which blocks of newly made RNA are put together, allowing one gene to produce different combinations of blocks and hence different proteins. In addition to its role as a splicing factor, Nova is the target in a neurodegenerative disease called paraneoplastic opsoclonus-myoclonus, which causes loss of control of the eyes and uncontrollable shaking.

“Previous work in our laboratory had revealed how Nova bound to RNA, and we had identified a few specific target RNAs in the brain,” says Darnell, Robert and Harriet Heilbrunn Professor at Rockefeller and Howard Hughes Medical Institute Investigator. “These studies showed that Nova was the first splicing factor in mammals that was restricted to a particular tissue. But what we really want to know is, what is the full array of RNAs that Nova binds to in the brain?”

When Darnell and his colleagues began sifting through the RNA sites their CLIP technique had identified, they found 18 that were located near small pieces of immature RNAs, near areas of alternatively spliced blocks of a number of new genes. They were able to verify that these tags were real Nova RNA splicing targets by comparing these RNAs in normal mice and mice lacking the Nova protein. “We’re finding that Nova is extremely important and maybe the single factor that is responsible for neuronal splicing for some targets,” Darnell says.

What’s more, the group of RNA targets identified by CLIP encode a common set of brain functions. “Looking at these targets as a group, they have a tremendous biological coherence to them,” says Darnell. “Almost 80 percent of the RNAs that come up repeatedly in CLIP encode a function related to the neuronal synapse.” Synapses are the point of contact between neurons, where information is exchanged in the form of electrical currents. “These findings suggest that Nova has evolved to regulate a set of RNAs that have a coordinate function. So, if you turn Nova function up or down, you’ll regulate a group of RNAs en masse.”

And there may be additional functions for Nova beyond RNA splicing. “CLIP opens a door to studying the function of Nova in the human brain,” says Jernej Ule, a graduate fellow in Darnell’s lab who carried out the research with co-first author Kirk B. Jensen, research assistant professor. “We can now better ask, for example, how can the loss of one RNA-binding protein lead to mental retardation in the Fragile X syndrome?”

December 12, 2003

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