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.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/627192 |
| Date | 17 January 2018 |
| Creators | Deng, Zhongyuan, Zhang, Shen, Gu, Shaohua, Ni, Xinzhi, Zeng, Wenxian, Li, Xianchun |
| Contributors | Univ Arizona, Dept Entomol, Univ Arizona, Inst BIO5 |
| Publisher | MDPI AG |
| Source Sets | University of Arizona |
| Language | English |
| Detected Language | English |
| Type | Article |
| Rights | © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. |
| Relation | http://www.mdpi.com/1422-0067/19/1/279 |
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