Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries.

Kajsa Fritzell,Claes Andréasson,Magdalena Otrocka,Li-Di Xu,Marie Öhman

doi:10.1093/nar/gky1228

Kajsa Fritzell, Claes Andréasson + Show 3 more

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https://doi.org/10.1093/nar/gky1228

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Abstract

Adenosine to inosine editing is common in the human transcriptome and changes of this essential activity is associated with disease. Children with ADAR1 mutations develop fatal Aicardi-Goutières syndrome characterized by aberrant interferon expression. In contrast, ADAR1 overexpression is associated with increased malignancy of breast, lung and liver cancer. ADAR1 silencing in breast cancer cells leads to increased apoptosis, suggesting an anti-apoptotic function that promotes cancer progression. Yet, suitable high-throughput editing assays are needed to efficiently screen chemical libraries for modifiers of ADAR1 activity. We describe the development of a bioluminescent reporter system that facilitates rapid and accurate determination of endogenous editing activity. The system is based on the highly sensitive and quantitative Nanoluciferase that is conditionally expressed upon reporter-transcript editing. Stably introduced into cancer cell lines, the system reports on elevated endogenous ADAR1 editing activity induced by interferon as well as knockdown of ADAR1 and ADAR2. In a single-well setup we used the reporter in HeLa cells to screen a small molecule library of 33 000 compounds. This yielded a primary hit rate of 0.9% at 70% inhibition of editing. Thus, we provide a key tool for high-throughput identification of modifiers of A-to-I editing activity in cancer cells.

Highlights

Editing of RNA by deamination of adenosines to inosines (A-to-I) is an essential process in mammals [1,2,3] that can be catalyzed by two enzymes, ADAR1 and ADAR2
A dual bioluminescence reporter sensitive to small changes in RNA editing activity We aimed at constructing a reporter to monitor editing activity in human cells that: (i) is sensitive to small fluctuations in editing activity, (ii) yields reproducible data points, (iii) has a quantitative readout suitable for high-throughput measurements and (iv) is sensitive to endogenous editing elicited by external stimuli or inhibitors
Editing of the reporter was verified by Sanger sequencing and shown to be greatly decreased after ADAR1 silencing, in line with the result from the reporter assay (Figure 4D). These results show that ADAR1 plays a major role in editing of the reporter RNA hairpin in HeLa cells and is consistent with previous evidence pointing to high ADAR1 activity in cancer cells [17,18,19,20]

Summary

Introduction

Editing of RNA by deamination of adenosines to inosines (A-to-I) is an essential process in mammals [1,2,3] that can be catalyzed by two enzymes, ADAR1 and ADAR2. The GluA2 transcript is highly edited at another site in exon 13 This editing event leads to changed translation form arginine to glycine (R/G). Several other genes involved in neurotransmission have been shown to utilize A-to-I editing to express alternative protein isoforms with functional consequences for receptor topology and assembly (reviewed in [4]). These transcripts are frequently edited by both ADAR1 and ADAR2, but some sites are enzyme specific [2,5,6]. Loss of ADAR1 in hematopoietic stem cells leads to an upregulation of interferon-stimulated genes (ISGs) [9]. Mutations in the ADAR1 gene cause Aicardi-Goutieres syndrome, which is a fatal autoimmune disease in children caused by an upregulation of ISGs [12]

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