活细胞内人AGTR1-3′UTR的荧光标记及应激定位分析  

A Fluorescence Labeling System for the 3′UTR of Homo Sapiens AGTR1 mRNA and Its Application in the Subcellular Localization Analysis Under Cell Stress

作　　者：张毅[1,2,3,4,5,6] 高星杰[3,4,5,6] 付雪[3,4,5,6] 苏超[3,4,5,6] 史雪彬[3,4,5,6] 段中潮[3,4,5,6] 付晓[3,4,5,6] 何津岩[3] 杨洁[3,4,5,6] 

机构地区：[1]天津医科大学药学院,天津300070 [2]天津市临床药物关键技术重点实验室,天津300070 [3]天津医科大学基础医学院,天津300070 [4]天津医科大学基础医学研究中心,天津300070 [5]天津市细胞与分子免疫学重点实验室,天津300070 [6]教育部免疫微环境与疫病重点实验室,天津市300070

出　　处：《中国细胞生物学学报》2014年第5期631-637,共7页Chinese Journal of Cell Biology

基　　金：国家杰出青年科学基金(批准号:31125012);国家自然科学基金(批准号:21305103;31100967;31170830;31370749);中国博士后科学基金(批准号:2013T60258)资助的课题~~

摘　　要：利用噬菌体衣壳蛋白MS2和带有序列特异性茎环结构(含有MS2蛋白结合位点)的RNA之间的高度亲和力,对外源性人血管紧张素1型受体(angiotensin II receptor type 1,AGTR1)mRNA 3′端非翻译区(3′untranslated region,3′UTR)片段进行红色荧光标记,进而在活细胞(HeLa)内研究该mRNA片段的应激生物学行为。通过在pSG5空载体质粒上先后插入两个双链DNA目的片段AGTR1-3′UTR和24×MS2,构建重组质粒pSG5/AGTR1-3′UTR/24×MS2,并将该质粒与重组质粒pERFP/MS2和pEGFP/C1-G3BP共转染入Hela细胞。荧光显微成像结果显示,AGTR1-3′UTR-24×MS2 mRNA片段能够携带具有入核信号的MS2-RFP融合蛋白离开胞核进入胞浆,而且在亚砷酸盐刺激下,红色荧光标记的AGTR1-3′UTR-24×MS2 mRNA片段可在胞浆中形成与应激蛋白G3BP-GFP共定位的颗粒。该结果表明,针对AGTR1-3′UTR片段的MS2-RFP荧光标记系统构建成功,该荧光标记系统能有效避免假阳性的荧光信号。在细胞受到氧化应激时,AGTR1-3′UTR会被招募至胞浆中的应激颗粒结构中,启示了AGTR1-3′UTR区域对于调控AGTR1 mRNA在细胞内的应激定位具有重要作用。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 AGTRI-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 AGTRI-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 A GTR1 mRNAs into stress granules was modulated by the 3＇UTR.

关 键 词：人血管紧张素1型受体 3’端非翻译区 MS2标记 重组质粒 荧光标记 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]