Study of circular RNA translation using reporter systems in living cells
Study of circular RNA translation using reporter systems in living cells
复制标题
使用活细胞报告系统研究环状RNA翻译
DOI：
10.1016/j.ymeth.2021.03.011
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发表时间：
2021
期刊：
影响因子：
4.8
通讯作者：
Wang Zefeng
中科院分区：
文献类型：
--
作者：
Chen Chuyun;Yang Yun;Wang Zefeng
近年来，在真核生物中发现了大量的环状RNA（circular RNA，circRNA），其中一些被报道是通过帽非依赖性方式翻译的。然而，对circRNA翻译的研究仍然不是微不足道的。在这里，我们描述了两个不同的系统，以产生可翻译的circRNA含有验证的开放阅读框架（ORF），以分析它们在活细胞中的翻译。第一个系统是含有单个外显子的质粒报告基因，该外显子具有以相反顺序分裂的GFP片段，其可以被有效地反向剪接以产生编码完整GFP的circRNA。第二个系统是一个自剪接报告基因，包含一个完整的海肾荧光素酶（Rluc）ORF和侧翼的分裂组I内含子以相反的顺序，它可以产生circRNA通过在体外自剪接的前体RNA。这两种circRNA系统均可作为circRNA翻译机制研究的平台，也可作为IRES介导的翻译活性检测的可靠系统。
Recently, a large number of circular RNAs (circRNAs) were discovered in eukaryotes, some of which were reported to be translated through a cap-independent fashion. However, study of circRNA translation is still not trivial. Here we describe two distinct systems to generate the translatable circRNAs containing validated open reading frames (ORF) to analyze their translation in living cells. The first system is a plasmid reporter containing a single exon with split GFP fragments in reverse order, which can be efficiently back-spliced to generate a circRNA encoding intact GFP. The second system is a self-splicing reporter containing an intact Renilla luciferase (Rluc) ORF and the flanking split group I introns in reverse order, which can produce circRNAs throughin vitroself-splicing of the precursor RNAs. Both circRNA systems can serve as the platforms for mechanistic studies of circRNA translation, and also serve as the reliable systems to measure the activity of IRES-mediated translation.