Useful Bicistronic Reporter System for Studying Poly(A) Site-Defining cis Elements and Regulation of Alternative Polyadenylation

文献类型： 外文期刊

第一作者： Zeng, Wenxian

作者： Zeng, Wenxian;Deng, Zhongyuan;Zhang, Shen;Gu, Shaohua;Li, Xianchun;Ni, Xinzhi;Li, Xianchun;Li, Xianchun

作者机构：

关键词： alternative polyadenylation;cis elements;bicistronic reporter system;human CD47;polyadenylation efficiency;synthetic polyadenylation sites

期刊名称：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES （ 影响因子：5.923； 五年影响因子：6.132 ）

ISSN： 1422-0067

年卷期： 2018 年 19 卷 1 期

页码：

收录情况： SCI

摘要： The link between polyadenylation (pA) and various biological, behavioral, and pathological events of eukaryotes underlines the need to develop in vivo polyadenylation assay methods for characterization of the cis-acting elements, trans-acting factors and environmental stimuli that affect polyadenylation efficiency and/or relative usage of two alternative polyadenylation (APA) sites. The current protein-based CAT or luciferase reporter systems can measure the polyadenylation efficiency of a single pA site or candidate cis element but not the choice of two APA sites. To address this issue, we developed a set of four new bicistronic reporter vectors that harbor either two luciferase or fluorescence protein open reading frames connected with one Internal Ribosome Entry Site (IRES). Transfection of single or dual insertion constructs of these vectors into mammalian cells demonstrated that they could be utilized not only to quantify the strength of a single candidate pA site or cis element, but also to accurately measure the relative usage of two APA sites at both the mRNA (qRT-PCR) and protein levels. This represents the first reporter system that can study polyadenylation efficiency of a single pA site or element and regulation of two APA sites at both the mRNA and protein levels.

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