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Technique may identify novel disease genes at a faster clip

ing implicated in human diseases. Thus, a technique that can help identify the multiple RNA targets regulated by an RNA-binding protein could potentially aid in understanding the cause of many human diseases.

To facilitate identification of the target proteins, the scientists adapted a technique that had been used in the test tube to identify the targets of RNA-binding proteins. This technique involved irradiating molecules with ultraviolet light, which caused a cross-linking reaction that chemically bonded the protein with its RNA target. The bond is so tight that the molecules could be isolated and identified together.

Darnell and his colleagues made some enhancements that resulted in the development of their "cross-linking and immunoprecipitation" (CLIP) technique. The researchers began by irradiating intact mouse brains with UV light, seeking to weld together RNA-binding proteins and their RNA targets in living tissue. Following a technically demanding purification and analytical procedure, the researchers were able to pinpoint some 340 Nova CLIP "tags" -- telltale pieces of Nova-bound RNA that identified the RNA target molecule and revealed where the Nova protein bound to it. The researchers verified that the tags represented functional Nova RNA targets by comparing their splicing in wild-type mice with knockout mice lacking Nova.

The striking splicing changes in the knockout mice, said Darnell, constituted proof that Nova is the central regulator of splicing in a whole set of RNA molecules found in the brain. "We're finding the Nova is an extremely important factor--maybe the factor--that is responsible for neuronal splicing for some targets," he said.

Their studies turned up another important observation: Nova does not act randomly. "Looking at these targets as a group, they have a tremendous biological coherence to them," said Darnell. "Almost seventy percent of them are RNAs that have something to do with the neuronal
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Contact: Jim Keeley
keeleyj@hhmi.org
301-215-8858
Howard Hughes Medical Institute
13-Nov-2003


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