Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Researchers describe new technique for cataloging RNA targets in rare brain disease

14.11.2003


RNA, often thought of as merely the chemical messenger that helps decode DNA’s genetic instructions for making proteins, can itself play a crucial role in regulating protein expression. Not surprisingly, this regulation occurs through proteins that bind to RNA. All cells in the body, especially nerve cells in the brain, use and regulate RNA in an exquisite fashion.



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.

Joseph Bonner | EurekAlert!
Further information:
http://www.rockefeller.edu/

More articles from Life Sciences:

nachricht Water world
20.11.2017 | Washington University in St. Louis

nachricht Carefully crafted light pulses control neuron activity
20.11.2017 | University of Illinois at Urbana-Champaign

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: A “cosmic snake” reveals the structure of remote galaxies

The formation of stars in distant galaxies is still largely unexplored. For the first time, astron-omers at the University of Geneva have now been able to closely observe a star system six billion light-years away. In doing so, they are confirming earlier simulations made by the University of Zurich. One special effect is made possible by the multiple reflections of images that run through the cosmos like a snake.

Today, astronomers have a pretty accurate idea of how stars were formed in the recent cosmic past. But do these laws also apply to older galaxies? For around a...

Im Focus: Visual intelligence is not the same as IQ

Just because someone is smart and well-motivated doesn't mean he or she can learn the visual skills needed to excel at tasks like matching fingerprints, interpreting medical X-rays, keeping track of aircraft on radar displays or forensic face matching.

That is the implication of a new study which shows for the first time that there is a broad range of differences in people's visual ability and that these...

Im Focus: Novel Nano-CT device creates high-resolution 3D-X-rays of tiny velvet worm legs

Computer Tomography (CT) is a standard procedure in hospitals, but so far, the technology has not been suitable for imaging extremely small objects. In PNAS, a team from the Technical University of Munich (TUM) describes a Nano-CT device that creates three-dimensional x-ray images at resolutions up to 100 nanometers. The first test application: Together with colleagues from the University of Kassel and Helmholtz-Zentrum Geesthacht the researchers analyzed the locomotory system of a velvet worm.

During a CT analysis, the object under investigation is x-rayed and a detector measures the respective amount of radiation absorbed from various angles....

Im Focus: Researchers Develop Data Bus for Quantum Computer

The quantum world is fragile; error correction codes are needed to protect the information stored in a quantum object from the deteriorating effects of noise. Quantum physicists in Innsbruck have developed a protocol to pass quantum information between differently encoded building blocks of a future quantum computer, such as processors and memories. Scientists may use this protocol in the future to build a data bus for quantum computers. The researchers have published their work in the journal Nature Communications.

Future quantum computers will be able to solve problems where conventional computers fail today. We are still far away from any large-scale implementation,...

Im Focus: Wrinkles give heat a jolt in pillared graphene

Rice University researchers test 3-D carbon nanostructures' thermal transport abilities

Pillared graphene would transfer heat better if the theoretical material had a few asymmetric junctions that caused wrinkles, according to Rice University...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Ecology Across Borders: International conference brings together 1,500 ecologists

15.11.2017 | Event News

Road into laboratory: Users discuss biaxial fatigue-testing for car and truck wheel

15.11.2017 | Event News

#Berlin5GWeek: The right network for Industry 4.0

30.10.2017 | Event News

 
Latest News

Antarctic landscape insights keep ice loss forecasts on the radar

20.11.2017 | Earth Sciences

Filling the gap: High-latitude volcanic eruptions also have global impact

20.11.2017 | Earth Sciences

Water world

20.11.2017 | Life Sciences

VideoLinks
B2B-VideoLinks
More VideoLinks >>>