A mammalian reporter system for fast and quantitative detection of intracellular A-to-I RNA editing levels.
An important molecular mechanism to create protein diversity from a limited set of genes is A-to-I RNA editing. RNA editing converts single adenosines into inosines in pre-mRNA. These single base conversions can have a wide variety of consequences. Editing can lead to codon changes and, consequently, altered protein function. Moreover, editing can alter splice sites and influences miRNA biogenesis and target recognition. The two enzymes responsible for editing in mammals are adenosine deaminase acting on RNA (ADAR) 1 and 2. However, it is currently largely unknown how the activity of these enzymes is regulated in vivo. Editing activity does not always correlate with ADAR expression levels, suggesting posttranscriptional or posttranslational mechanisms for controlling activity. To investigate how editing is regulated in mammalian cells, we have developed a straightforward quantitative reporter system to detect editing levels. By employing luciferase activity as a readout, we could easily detect different levels of editing in a cellular context. In addition, increased levels of ADAR2 correlated with increased levels of luciferase activity. This reporter system therefore sets the stage for the effective screening of cDNA libraries or small molecules for strong modulators of intracellular editing to ultimately elucidate how A-to-I editing is regulated in vivo.
Gommans
Willemijn M
WM
Department of Biological Sciences, 111 Research Drive, Iacocca Hall D226, Bethlehem, PA 18015-4732, USA.
McCane
Jill
J
Nacarelli
Gregory S
GS
Maas
Stefan
S
D000241:Adenosine / Q000378:metabolism; D000243:Adenosine Deaminase / Q000378:metabolism*; D000595:Amino Acid Sequence; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007288:Inosine / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism; D008969:Molecular Sequence Data; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D017393:RNA Editing; D016601:RNA-Binding Proteins
D035683:MicroRNAs; D016601:RNA-Binding Proteins; D007288:Inosine; D012313:RNA; D008156:Luciferases; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase; D000241:Adenosine