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RNA Biol. 2013 Feb;10(2):192-204. doi: 10.4161/rna.23208. Epub 2013 Jan 25.

A high-throughput screen to identify enhancers of ADAR-mediated RNA-editing.

Author information

  • 1Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

Abstract

Adenosine to inosine deamination of RNA is widespread in metazoa. Inosines are recognized as guanosines and, therefore, this RNA-editing can influence the coding potential, localization and stability of RNAs. Therefore, RNA editing contributes to the diversification of the transcriptome in a flexible manner. The editing reaction is performed by adenosine deaminases that act on RNA (ADARs), which are essential for normal life and development in many organisms. Changes in editing levels are observed during development but also in neurological pathologies like schizophrenia, depression or tumors. Frequently, changes in editing levels are not reflected by changes in ADAR levels suggesting a regulation of enzyme activity. Until now, only a few factors are known that influence the activity of ADARs. Here we present a two-stage in vivo editing screen aimed to isolate enhancers of editing. A primary, high-throughput yeast-screen is combined with a more accurate secondary screen in mammalian cells that uses a fluorescent read-out to detect minor differences in RNA-editing. The screen was successfully employed to identify DSS1/SHFM1, the RNA binding protein hnRNP A2/B1 and a 3' UTR as enhancers of editing. By varying intracellular DSS1/SHFM1 levels, we can modulate A to I editing by up to 30%. Proteomic analysis indicates an interaction of DSS1/SHFM1 and hnRNP A2/B1 suggesting that both factors may act by altering the cellular RNP landscape. An extension of this screen to cDNAs from different tissues or developmental stages may prove useful for the identification of additional enhancers of RNA-editing.

KEYWORDS:

DSS1; RNA-binding; RNA-editing; TREX-2; regulation of RNA-editing; yeast screen

PMID:
23353575
[PubMed - indexed for MEDLINE]
PMCID:
PMC3594278
Free PMC Article

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