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Int. J. Mol. Sci. 2018, 19(1), 279; https://doi.org/10.3390/ijms19010279

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

1
College of Animal Science and Technology, Northwest A&F University, No. 22 Xinong Road, Yangling 712100, Shaanxi, China
2
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
3
United States Department of Agriculture, Agricultural Research Service, Crop Genetics and Breeding Research Unit, Tifton, GA 31793, USA
4
Department of Entomology and BIO5 Institute, University of Arizona, Tucson, AZ 85721, USA
*
Authors to whom correspondence should be addressed.
Received: 13 December 2017 / Revised: 5 January 2018 / Accepted: 15 January 2018 / Published: 17 January 2018
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

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. View Full-Text
Keywords: alternative polyadenylation; cis elements; bicistronic reporter system; human CD47; polyadenylation efficiency; synthetic polyadenylation sites alternative polyadenylation; cis elements; bicistronic reporter system; human CD47; polyadenylation efficiency; synthetic polyadenylation sites
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Deng, Z.; Zhang, S.; Gu, S.; Ni, X.; Zeng, W.; Li, X. Useful Bicistronic Reporter System for Studying Poly(A) Site-Defining cis Elements and Regulation of Alternative Polyadenylation. Int. J. Mol. Sci. 2018, 19, 279.

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