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A radar for ADAR: Altered gene tracks RNA editing in neurons
Date:12/25/2011

requiring ADAR to fix a purposely broken individual letter of RNA on an engineered gene.

"We're actually repairing RNA at the level of a single informational bit, or nucleotide," Reenan said. "Here we've shown we can take a mutant version of a gene and restore its function, but at the level of RNA rather than DNA."

A reporter of an editor

Reenan and third author Kyle Jay began working to create the reporter in 2006 when Jay was an undergraduate student just embarking on what would become a celebrated senior thesis at Brown. They started with a well-known tool of molecular biology: a jellyfish gene that produces a protein that glows green upon exposure to ultraviolet light. The strategy was to intentionally break the gene in a way that ADAR is uniquely suited to fix.

First they engineered the gene to include necessary "intron" code that requires a specific splicing operation to take place. Then they inserted the "stop codon" T-A-G in place of T-G-G, which causes transcription to cease, effectively preventing production of the green fluorescent protein. But before splicing occurs and when ADAR finds the stop codon U-A-G in the RNA transcript, it edits the A to an I, which restores the correct information, and translation of the whole gene proceeds as if there were no stop mutation in the DNA. So when splicing and ADAR editing occurs, neurons with the gene reporter glow green.

To see where ADAR editing and splicing were occurring, compared to just splicing alone, they also rigged up an engineered gene with the splicing requirement, but not the T-A-G codon. That would produce yellow fluorescent protein when splicing alone occurred.

Armed with their new ADAR reporter, Reenan and lead author James Jepson set out to make some biological observations in flies. One was that ADAR activity is more pronounced in certain parts of the brains of developing larvae than it is in the brains of adults. The team also found w
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Contact: David Orenstein
david_orenstein@brown.edu
401-863-1862
Brown University
Source:Eurekalert  

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