RNA Map Provides First Comprehensive Understanding Of Alternative Splicing

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RNA Map Provides First Comprehensive Understanding Of Alternative

Splicing

http://www.medicalnewstoday.com/medicalnews.php?newsid=56299

It's biology's version of the director's cut. In much the same way

that numerous films could be stitched together from a single reel of

raw footage, a molecular process called alternative splicing enables

a single gene to produce multiple proteins. Now a new RNA map,

created by a team of researchers at Rockefeller University and the

Medical Institute and announced in the journal Nature,

shows for the first time how the specific location of short snippets

of RNA affects the way that alternative splicing is controlled in

the brain.

Though scientists have begun to appreciate how alternative splicing

adds a layer of complexity to brain processes that enable us to

think and learn, exactly how alternative splicing is regulated

during these processes -- and in some cases is uncontrolled (or

dysregulated) to cause disease -- has remained elusive. The map

provides the first comprehensive understanding of how alternative

splicing works throughout the genome. The results have implications

for a better understanding of such brain functions as learning and

memory, neurological diseases and cancer biology.

" This finding is a significant advance in our understanding of

splicing, and it suggests that it will be possible to understand how

different splicing factors weave together to regulate complex

patterns of genes, which in turn is relevant to generating

complexity of function, " says senior author Darnell,

professor and head of the Laboratory of Molecular Neuro-Oncology at

Rockefeller and investigator at HHMI.

RNA splicing is the process by which the initial RNA copy of a 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. The exon can

be spliced in or out in a binary, computer-like fashion. By

regulating alternative splicing cells can produce a wide variety of

proteins from a finite number of genes. This capacity is believed to

be critical to the complex workings of human cells such as those

found in the neurons of the brain.

The researchers focused on a brain protein that binds to RNA called

Nova. Darnell and his colleagues first identified Nova in 1993 as

the target protein in a neurodegenerative disease termed POMA

(paraneoplastic opsoclonus-myoclonus ataxia) that is also associated

with several types of cancers. Since then the laboratory has focused

on identifying RNA sequences -- and in particular, identifying

alternatively spliced pre-mRNAs -- that Nova binds to. In the last

three years, in work published in Science and Nature Genetics, the

Darnell lab identified over 50 Nova-regulated alternatively spliced

exons, using new techniques developed at Rockefeller specifically to

find Nova RNA targets, and validating their results in " knockout "

mice that were missing Nova.

In the new study, Darnell, with co-first authors Jernej Ule and

Giovanni Stefani, took these 50 RNA transcripts and searched them

for clusters of sequences they had previously identified as Nova

binding sites through biochemical and, in collaboration with former

Rockefeller University structural biologist Burley, X-ray

crystallographic studies. Unexpectedly, this search revealed four

discrete peaks where the binding clusters locate. Furthermore, the

location of the peaks correlated with Nova's action on regulating

whether the alternative exon is spliced in or out.

The researchers tested whether this RNA map was valid by asking

whether it could predict how Nova would act on RNA transcripts that

had yet to be discovered. They took a bioinformatics approach, using

a database of all alternatively spliced RNAs compiled by co-authors

Terry Gaasterland and Bahar Taneri, to search for new genes that had

clusters of Nova binding sites. Of the 50 or so transcripts with

such clusters, 30 turned out to be alternatively spliced in a Nova-

dependent way. Of those, all 30 fit the rules of the RNA map.

" In other words, every transcript that we could predict as a Nova-

regulated alternatively spliced RNA fit the prediction of this map, "

says Darnell. " Half of them were inhibited by Nova and half were

enhanced in their exon use by Nova, and every one very cleanly fit

the pattern. "

The researchers also simulated alternative splicing in the test

tube, mixing purified RNA and a splicing extract. When purified Nova

was added to the extracts, it bound to the mRNA clusters, altering

the outcome of the how the splicing machinery was able to assemble

in a manner that again conformed to the predictions of the RNA map.

In one case Nova blocked specific components of the splicing

machinery, in another it enhanced the ability of this machinery to

assemble the right way and use an alternatively spliced site that is

otherwise poorly utilized.

By offering a global understanding of how alternative splicing works

across the genome, the map has implications for the treatment of a

growing list of human neurologic diseases in which RNA regulation,

and particularly RNA splicing, has been implicated as the primary

cause, including certain types of cancer and a number of brain and

muscle disorders.

" Given that the complexity of the brain is orders and orders of

magnitude more complex than the number of genes we have, one of the

intriguing things about alternative splicing is that a relatively

small number of regulatory splicing factors acting in concert on a

single transcript can potentially generate a large number of

different protein variants, " says Darnell.

" There is a converging set of observations indicating that as

neurologic diseases are better understood, alternative splicing is

going to play an important role in generation of disease and

therefore an important role in normal generation of cognitive

function, " he adds. " Our new work lays out an approach to developing

a global understanding of how alternative splicing is regulated by

one disease-associated protein, Nova, offering a route by which

scientists may now be able to approach a number of diseases with a

fresh start. "

Finally, Darnell's work has shown that Nova is expressed in certain

types of cancer cells. Cancer cells operate by dysregulating gene

expression, and Darnell believes that further studies are needed to

determine whether Nova is a cause of dysregulated gene expression at

the level of alternative splicing in a cancer cell.

" The right splicing factor in the wrong environment could wreck

havoc and change the quality of proteins in a tumor cell, " he says.