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

Zhongyuan Deng, Shen Zhang, Shaohua Gu, Xinzhi Ni, Wenxian Zeng, Xianchun Li

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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.

Original languageEnglish (US)
Article number279
JournalInternational journal of molecular sciences
Volume19
Issue number1
DOIs
StatePublished - Jan 17 2018

Keywords

  • Alternative polyadenylation
  • Bicistronic reporter system
  • Cis elements
  • Human CD47
  • Polyadenylation efficiency
  • Synthetic polyadenylation sites

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

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