A mammalian reporter system for fast and quantitative detection of intracellular A-to-I RNA editing levels

Abstract

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 post-transcriptional or post-translational mechanisms for controlling activity. To investigate how editing is regulated in mammalian cells, we have developed a straight-forward quantitative reporter system to detect editing levels. By employing luciferase activity as a read-out, 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.

A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1

“…To date, reporter plasmids containing GluR B sequence as the substrate have been used (20–22, 24, 25). Although GluR B-derived reporter plasmids work efficiently for ADAR2, they fail with ADAR1 (24, 25). …”

Reduced adenosine-to-inosine miR-455-5p editing promotes melanoma growth and metastasis

“…The ADAR1 A-to-I RNA editing hairpin loop luciferase reporter was used as described previously 35 The ADAR1 promoter luciferase assay was performed as follows. The ADAR1 promoter region (nucleotides −1000 to +65 from the transcription initiation site) was amplified from C8161 genomic DNA using the following primers, Forward: 5′- GGGGTACCAGCCTCGGTTTCTACACCTGC-3′; Reverse: 5′- CCGCTCGAGGGTTCAATTTCGCTTTCGTTTC-3′.…”

Comparison of RNA Editing Activity of APOBEC1-A1CF and APOBEC1-RBM47 Complexes Reconstituted in HEK293T Cells

“…Investigation of RNA deamination is difficult when compared to similar enzymatic editing activity on other polynucleotides, as there are few systems that can effectively provide a readout for when RNA editing is occurring. A few have been proposed for adenosine deamination [28][29][30], and some have been adapted for cytidine deamination [6,31,32], although these can sometimes be time consuming, show low sensitivity, or are difficult to quantify precisely. Refining on several of these methods led to our development of a system where RNA editing directly induces a shift in subcellular localization of an engineered eGFP [33] construct.…”