Useful bicistronic reporter system for studying poly (A) site-defining cis elements and regulation of alternative polyadenylation

Authors: DENG, Z - Northwest Agricultural & Forestry University; ZHANG, S - Chinese Academy Of Agricultural Sciences; GU, S - Chinese Academy Of Agricultural Sciences; Ni, Xinzhi; ZENG, W - Northwest Agricultural & Forestry University; LI, X - University Of Arizona

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2018
Publication Date: 1/17/2018
Citation: Deng, Z., Zhang, S., Gu, S., Ni, X., Zeng, W., Li, X. 2018. Useful bicistronic reporter system for studying poly (A) site-defining cis elements and regulation of alternative polyadenylation. International Journal of Molecular Sciences. 19(1):Article 279.

Interpretive Summary: Polyadenylation is the addition of a poly adenine phosphate tail to a messenger RNA, which is part of the process that produces mature messenger RNA for translation, and an essential step of gene/transposon regulation that directs all biological and behavioral events of eukaryotes. The link between polyadenylation and various biological, behavioral and pathological events of eukaryotes underlines the need to develop in vivo polyadenylation assay methods for characterization of various regulatory sequence elements, protein factors and environmental stimuli that affect polyadenylation efficiency and/or relative usage of two alternative polyadenylation sites. The current protein-based reporter systems can measure the polyadenylation efficiency of a single polyadenylation site or candidate cis element but not the choice of two alternative polyadenylation sites. To address this issue, we developed a set of four new reporter vectors that harbor either two luciferase or fluorescence proteins. Transfection of single or dual insertion constructs of these vectors into mammalian cells demonstrated that the vectors could be utilized not only to quantify the strength of a single candidate polyadenylation site or cis-regulatory element, but also accurately measure the relative usage of two alternative polyadenylation sites at both the mRNA and protein levels. This research represents the first reporter system that can study polyadenylation efficiency of a single polyadenylation site or cis-regulatory element, and regulation of two APA sites at both the mRNA and protein levels.

Technical 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 regulatory 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 system 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 dicistronic 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-regulatory element, but also 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 cis-regulatory element, and regulation of two APA sites at both the mRNA and protein levels.