TY - JOUR
T1 - Useful bicistronic reporter system for studying Poly(A) site-defining cis elements and regulation of alternative polyadenylation
AU - Deng, Zhongyuan
AU - Zhang, Shen
AU - Gu, Shaohua
AU - Ni, Xinzhi
AU - Zeng, Wenxian
AU - Li, Xianchun
N1 - Funding Information: Acknowledgments: This work was supported by United States Department of Agriculture, National Institute of Food and Agriculture (hatch grant ARZT-1360890-H31-164 and multi state grant ARZT-1370400-R31-168), Beijing Nova Program (Z15110000 03150118), Beijing talents fund (2015000021223ZK29) and National Natural Science Foundation of China (31772164 and 31401737). Publisher Copyright: © 2018 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2018/1/17
Y1 - 2018/1/17
N2 - 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.
AB - 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.
KW - Alternative polyadenylation
KW - Bicistronic reporter system
KW - Cis elements
KW - Human CD47
KW - Polyadenylation efficiency
KW - Synthetic polyadenylation sites
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U2 - 10.3390/ijms19010279
DO - 10.3390/ijms19010279
M3 - Article
C2 - 29342112
SN - 1661-6596
VL - 19
JO - International journal of molecular sciences
JF - International journal of molecular sciences
IS - 1
M1 - 279
ER -