Useful Bicistronic Reporter System for Studying Poly(A) Site-Defining cis Elements and Regulation of Alternative Polyadenylation
AffiliationUniv Arizona, Dept Entomol
Univ Arizona, Inst BIO5
bicistronic reporter system
synthetic polyadenylation sites
MetadataShow full item record
CitationUseful Bicistronic Reporter System for Studying Poly(A) Site-Defining cis Elements and Regulation of Alternative Polyadenylation 2018, 19 (1):279 International Journal of Molecular Sciences
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AbstractThe 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.
NoteOpen Access Journal.
VersionFinal published version
SponsorsUnited States Department of Agriculture; National Institute of Food and Agriculture [ARZT-1360890-H31-164, ARZT-1370400-R31-168]; Beijing Nova Program [Z15110000 03150118]; Beijing talents fund [2015000021223ZK29]; National Natural Science Foundation of China [31772164, 31401737]