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A Fluorescence Labeling System for the 3′UTR of Homo Sapiens AGTR1 mRNA and Its Application in the Subcellular Localization Analysis Under Cell Stress
Zhang Yi1,2,3,4,5,6#, Gao Xingjie3,4,5,6#, Fu Xue3-6, Su Chao3,4,5,6, Shi Xuebin3,4,5,6, Duan Zhongchao3,4,5,6, Fu Xiao3,4,5,6, He Jinyan3, Yang Jie3,4,5,6*
1College of Pharmacy, Tianjin Medical University, Tianjin 300070, China; 2Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), Tianjin 300070, China; 3Basic Medical Coll
Abstract: A MS2-tagged fluorescence labeling system for Homo sapiens angiotensin II receptor type 1 (AGTR1) 3′ end untranslated region (3′UTR) was constructed， and the system was further applied to visualize the subcellular localization of AGTR1-3′UTR under stress. The labeling system utilized the strong affinity between sequence-specific RNA stem-loops (MS2 protein binding sites) and the bacteriophage capsid protein MS2. The recombinant plasmids pSG5/AGTR1-3′UTR/24×MS2， which could be transcribed into AGTR1-3′UTR-24×MS2 mRNAs but not further into proteins in Hela cells， were constructed by being successively inserted into two dsDNA fragments AGTR1-3′UTR and 24×MS2， and further verified by enzyme digestion and order-checking. Then the plasmids were co-transfected with recombinant plasmids pERFP/MS2 and pEGFP/C1-G3BP into Hela cells. According to the results from fluorescence microscopy， AGTR1-3′UTR-24×MS2 mRNAs were found in cytoplasm together with the fusion protein RFP-MS2 that contained a nuclear localization signal sequence. The red fluorescence from MS2-tagged AGTR1-3′UTR was found highly co-localized with the green fluorescence from GFP-G3BP (a marker of stress granules under cell stress induced by arsenite). The results demonstrated that a fluorescence labeling system for Homo sapiens AGTR1-3′UTR was constructed successfully and expressed effectively. The subcellular co-localization of G3BP and AGTR1-3′UTR in stress granules indicated that the entrance of AGTR1 mRNAs into stress granules was modulated by the 3′UTR.
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