Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries
Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries
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DOI：
10.1093/nar/gky1228
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发表时间：
2019-02-28
影响因子：
14.9
通讯作者：
Ohman, Marie
中科院分区：
文献类型：
--
作者：
Fritzell, Kajsa;Xu, Li-Di;Ohman, Marie
腺苷到肌苷的编辑在人类转录组中很常见，这种基本活性的变化与疾病有关。ADAR 1突变的儿童发生以干扰素异常表达为特征的致命性Aicardi-Goutieres综合征。相反，ADAR 1过表达与乳腺癌、肺癌和肝癌的恶性程度增加相关。乳腺癌细胞中的ADAR 1沉默导致细胞凋亡增加，表明促进癌症进展的抗细胞凋亡功能。然而，需要合适的高通量编辑测定来有效地筛选化学文库中的ADAR 1活性修饰剂。我们描述了一种生物发光报告系统的发展，有利于快速，准确地确定内源性编辑活动。该系统是基于高灵敏度和定量的纳米荧光素酶，其在转录本编辑后有条件地表达。该系统稳定地引入癌细胞系中，报告了干扰素诱导的内源性ADAR 1编辑活性升高以及ADAR 1和ADAR 2的敲低。在单孔装置中，我们使用HeLa细胞中的报告基因筛选了33000种化合物的小分子文库。这在70%的编辑抑制下产生0.9%的主要命中率。因此，我们提供了一个关键的工具，用于高通量鉴定癌细胞中A-to-I编辑活性的修饰剂。
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-Goutieres 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.