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.
Highlights
Polyadenylation is an essential step of gene regulation that directs all biological and behavioral events of eukaryotes [1,2,3]
Sequencing of the positive clones verified that we successfully generated a bicistronic reporter system composed of two dual fluorescence (Figure 1A) and two dual luciferase (Figure 1B) reporter vectors by the seamless cloning strategy
The four vectors differ in their reporter proteins (fluorescence protein DsRed plus EGFP vs. humanized firefly plus renilla luciferase) and promoters that drive the transcription of the reporter genes
Summary
Polyadenylation is an essential step of gene regulation that directs all biological and behavioral events of eukaryotes [1,2,3] This transcription-coupled process is initiated by recognition of a pA site in the pre-mRNA, followed by termination of transcription, endonucleolytic cleavage of the pre-mRNA, and addition of a non-templated pA tail [1,2]. Among the several characterized auxiliary cis elements are the upstream U-rich elements [13], UGUA element [14,15], and US1A [12] as well as the downstream G-rich elements [16] and DS1A [12]. Many more auxiliary cis elements are yet to be identified
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