Scientists have previously shown that defects in RNA binding underlie several human brain disorders, but their RNA targets have been a mystery. Researchers at Rockefeller University have now developed a method that allows scientists, for the first time, to develop complete lists of RNAs that are regulated by RNA binding proteins.
According to the researchers, the method will generally be useful for scientists studying other diseases that result from defects in RNA regulation, including several autoimmune diseases, spinal muscular atrophy, and Fragile X mental retardation.
Reporting in the Nov. 14 issue of the journal Science, a team of scientists led by Robert B. Darnell, M.D., Ph.D., a professor at Rockefeller and an investigator at the Howard Hughes Medical Institute, showed that their new technique, called CLIP, can rapidly identify all the RNAs that bind to a protein that has been linked to the brain disorder POMA, or paraneoplastic opsoclonus myoclonus ataxia. These experiments were able to show that a protein called Nova plays a critical role in regulating alternative splicing within the brain.
"We have developed and validated a new methodology we term CLIP to help scientists interested in the role of RNA binding proteins in biology and disease," says Darnell. "We used CLIP to show that an RNA-binding protein called Nova regulates a biologically coherent -- that is, not a random -- set of RNAs whose proteins function at the synapse of nerve cells in the brain. This finding may help us better understand and treat the variety of diseases that involve the misregulation of RNA-binding proteins."
The scientists first became interested in Nova because the protein plays a key role in one of the paraneoplastic neurological disorders (PNDs) that the Darnell lab studies. Patients suffering from the PND termed POMA are unable to inhibit movement and suffer uncontrollable shaking. PNDs develop when cancer cells in the body prompt a tumor immune response that makes its way across the blood-brain barrier and disrupts the normal function of brain cells.
The brain is known as an "immune-privileged" site, meaning that proteins expressed only in the brain are not screened by the immune system as it goes through the process of learning which proteins are "self" and which are "foreign."
When a brain protein is expressed in a tumor elsewhere in the body, the immune system sees it as a foreign protein and mounts a strong response against it. This immune response, while good for eliminating the tumor, sometimes makes its way into the brain where it can attack those neurons that express the protein. In the case of POMA, the protein is Nova.
The exact nature by which the immune system attacks the brain is unclear, but POMA patients have high levels of antibodies against Nova in their spinal fluid. These antibodies bind a segment of Nova called the KH domain and inhibit Nova's interaction with RNA. Researchers suspect that the profound dysfunction of body movement that afflicts people with POMA is caused at least in part by a direct inhibition of RNA binding by Nova antibodies.
In 2000, Darnell and his colleagues, including Kirk B. Jensen, Ph.D., co-first author of the new Science paper, provided the first evidence that Nova is responsible for regulating RNA splicing in nerve cells.
RNA splicing is the process by which the initial RNA copy of any gene, known as pre-mRNA, is pieced together to produce a mature mRNA that codes for cellular proteins. In alternative splicing, different pieces of this pre-mRNA, called exons, are stitched together to produce different mRNAs, and thus different proteins. By regulating alternative splicing cells can produce a wide variety of proteins from a finite number of genes. This capacity is believed to critical to the complex workings of human cells such as those found in the neurons of the brain.
Nova was the first RNA binding protein discovered to regulate alternative splicing specifically in the brain.
"The finding that Nova regulates RNA splicing in neurons was satisfying as a proof of principle and raised interesting questions about Nova's function in disease," says Darnell. "More generally for Nova, and for all RNA-binding proteins that are important in biology and human disease, what we really want to know is what is the full array of RNAs that these proteins bind to -- the complete list."
To compile the complete list of RNAs to which Nova binds, Jensen and co-first author Jernej Ule combined a pair of standard techniques in the arsenal of biochemists -- photo cross-linking and immunoprecipitation -- with the use of brain tissue and a number of special techniques to create a new method called CLIP.
CLIP works by exposing cells to ultraviolet (UV) light, which causes very strong chemical bonds -- "irreversible links," says Jensen --to develop between RNA binding proteins and any RNAs with which they are in direct contact. After exposing mouse brain to UV light, the scientists then used a technique called immunoprecipitation, which uses antibodies to purify the samples. In the Science study, the Rockefeller scientists took serum containing Nova antibodies from POMA patients studied at The Rockefeller University Hospital for the immunoprecipitation step.
Using CLIP, the researchers identified an astounding set of 340 Nova RNA "tags": short stretches of RNA that harbor binding sites for Nova. By comparison, research using previously available methods, involving work done over the last seven years, had resulted in three RNA targets of Nova.
Darnell and colleagues focused on 18 CLIP tags that were adjacent to alternatively spliced exons. Of these, seven CLIP tags correctly identified exons where alternative splicing was misregulated in nerve cells in the brains of Nova knockout mice. These laboratory animals lack the gene for the Nova protein.
"Our data suggest that the neuron-specific protein Nova is the predominant -- and perhaps only -- factor necessary for mediating neuron-specific alternative splicing of a number of very interesting RNAs," says Darnell.
"CLIP opens a door to studying the function of Nova in the brain," says co-first author Jernej Ule, a graduate fellow in the Darnell lab.
"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?" Darnell says.
In addition to Darnell, Ule and Jensen, co-authors of the Science paper are Matteo Ruggio and Aldo Mele at Rockefeller, and Aljaz Ule at CREED, University of Amsterdam, Netherlands.
This research was supported by the National Institutes of Health, the Howard Hughes Medical Institute, Human Frontiers Science Program and the Cancer Research Institute.