Useful Bicistronic Reporter System for Studying Poly(A) Site-Defining cis Elements and Regulation of Alternative Polyadenylation

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.

Sequence determinants of polyadenylation-mediated regulation

“…The scale of our library allowed us to assay thousands of designed and native sequences from viral and human genomes. Our results are in line with previous studies, as in the case of base frequencies surrounding the cleavage site (Wang et al 2018) and the effect of the hexamer motif on expression (Deng et al 2018). Minor differences in the local base frequencies and the ranking of hexamer mutants may be attributed to the differences in assayed PASs and specific methods used.…”

A nuclear role for ARGONAUTE-2 in regulation of neuronal alternative polyadenylation

“…Construction of the Luciferase-based APA reporter vector was performed based on (Deng et al, 2018) using Restriction-Free Cloning (Unger et al, 2010)…”

“…Next, we quantified the relative usage of two Ret APA cis-regulatory sequences in response to AGO2 levels, by using a bicistronic luciferase-reporter, similar to the one reported in (Deng et al, 2018). The reporter, which harbors the APA cis-regulatory sequences of Ret mRNA 3ʹ, excludes many other potential regulatory elements that may reside on the transcript.…”

“…Construction of the Luciferase-based APA reporter vector was performed based on (Deng et al, 2018) using Restriction-Free Cloning (Unger et al, 2010) upon the psiCHECK-2 dual luciferase assay vector. All amplification reactions were performed using Q5 Hot Start High-Fidelity DNA polymerase (NEB, M0493S) in a LabCycler thermocycler (SensoQuest).…”

Identification and Characterization of the Masculinizing Function of the Helicoverpa armigera Masc Gene

“…Each developmental stage or body part had three independent biological replicates; RT-qPCR analysis of each biological replicate was repeated three times. The normalized expressions of HaMasc in each developmental stage or body part were calculated using the 2 −∆∆Ct method [59] 1) and one negative control siRNA (NC siRNA) were designed and synthesized by Ribobio Biotech (Guangzhou, China) to knock down the endogenous HaMasc in H. armigera larvae and embryo cell line QB-Ha-E-1 [60], respectively. QB-Ha-E-1 cells were routinely cultured at 28 • C with Grace's Insect Medium (Gibco/Life Technologies, New York, NY, USA) supplemented with a 10% fetal bovine serum (Gibco/Life Technologies, New York, NY, USA), 50 U/mL penicillin, and 50 µg/mL streptomycin (HyClone, Thermo Scientific, Logan, UT, USA).…”