A dual-luciferase reporter system for characterization of small RNA target gene in both mammalian and insect cells

Authors: DENG, ZHONGYUAN - Zhengzhou University; ZHANG, YUTING - Zhengzhou University; LI, LEYAO - Zhengzhou University; XIE, XINGCHENG - Chinese Academy Of Agricultural Sciences; ZHANG, MIN - Zhengzhou University; Ni, Xinzhi; LI, XIANCHUN - University Of Arizona

Submitted to: Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/16/2021
Publication Date: 7/7/2021
Citation: Deng, Z., Zhang, Y., Li, L., Xie, X., Zhang, M., Ni, X., Li, X. 2021. A dual-luciferase reporter system for characterization of small RNA target gene in both mammalian and insect cells. Insect Science. 29:631-644. https://doi.org/10.1111/1744-7917.12945.
DOI: https://doi.org/10.1111/1744-7917.12945

Interpretive Summary: Small non-coding RNAs (or small RNAs) are important regulatory molecules for gene expression in living organisms. They can be divided into microRNAs, small interfering RNAs, piwi-interacting RNAs, and transfer RNA-derived small RNAs based on their biogenesis, sequence features, and functions. MicroRNAs are involved in many cellular processes, including cell proliferation, differentiation, death, apoptosis, metabolism, and development in insects, as well as in mammals. Functions of microRNAs mainly achieve through the complicated binding process with their target genes. Nevertheless, binding of microRNAs to a target gene requires a minimum of only eight nucleotides of sequence complementarity. Computational prediction and luciferase reporter systems have been commonly used in identifying the interaction between microRNAs and their target genes. However, computational prediction produces high false positive rate. Thus, instead of depending on computational analysis, small RNA research is heavily relying on experimental confirmation, especially the classical luciferase reporter system. While there are several single or dual-luciferase reporter systems available for verifying target gene of small RNAs, they often work well in mammalian cell lines but poor in insect cell lines. In this study, two new dual-luciferase reporter vectors were developed to study small RNA-target gene relationships in RNAs in both insect and mammalian cell lines. Test application of the two new reporter vectors in two mammalian and four insect cell lines confirmed that one vector worked well in insect cell lines, and the other one performed well in both insect and mammalian cell lines. The two new vectors also revealed the presence of an unidentified endogenous factor that down-regulates male determination gene in the corn earworm ovary cell line but not in the fat body cell line. The use of the two dual-luciferase reporter systems could accelerate small RNA study in cell lines of pest (such as, fall armyworm and corn earworm) and other economically important beneficial insects (such as, the silkworm), which in turn may lead to the development of novel pest management tactics, or the improvement of beneficial insect production in general.

Technical Abstract: MicroRNAs (miRNAs) are regulatory RNA molecules that bind to target messenger RNAs (mRNAs) and affect the stability or translational efficiency of the bound mRNAs. Single or dual-luciferase reporter systems have been successfully used to identify miRNA target genes in mammalian cells. These reporter systems, however, are not sensitive enough to verify miRNA-target gene relationships in insect cell lines because the promoters of the target luciferase (usually Renilla) used in these reporter systems are too weak to drive sufficient expression of the target luciferase in insect cells. In this study, we replaced the SV40 promoter in the psiCHECK-2 reporter vector, which is widely used with mammalian cell lines, with the HSV-TK or AC5.1 promoter to yield two new dual-luciferase reporter vectors, designated psiCHECK-2-TK and psiCHECK-2-AC5.1, respectively. Only psiCHECK-2 and psiCHECK-2-AC5.1 had suitable target (Renilla)/reference (firefly) luciferase activity ratios in mammalian (HeLa and HEK293) and insect (Sf9, S2, Helicoverpa zea fat body and ovary) cell lines, while psiCHECK-2-TK had suitable Renilla/firefly luciferase activity ratios regardless of the cell line. Moreover, psiCHECK-2-TK successfully detected the interaction between Helicoverpa armigera miRNA9a and its target, the 3'-untranslated region of heat shock protein 90, in both mammalian and H. zea cell lines, but psiCHECK-2 failed to do so in H. zea cell lines. Furthermore, psiCHECK-2-TK with the target sequence, HzMasc (H. zea Masculinizer), accurately differentiated between H. zea cell lines with or without the negative regulation factor (miRNA or piRNA) of HzMasc. These data demonstrate that psiCHECK-2-TK can be used to functionally characterize small RNA target genes in both mammalian and insect cells.