转录激活因子1(ATF1)内部核糖体进入位点的鉴定及活性分析  

Structural and Functional Analyses of the Core Internal Ribosome Entry Site region of ATF1 5'-UTR

作　　者：李琪[1] 高文青[1] 朱瑞宇[1] 金坚[1] 

机构地区：[1]江南大学药学院分子药理实验室,江苏无锡214122

出　　处：《中国生物化学与分子生物学报》2015年第9期937-943,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.81101667);江苏省自然科学基金(No.BK2009071)~~

摘　　要：蛋白质翻译起始通常有两种机制,一是依赖帽结构的翻译,另一种是依赖5'非翻译区的内部核糖体进入位点(IRES).在后一种方式中,在某些IRES反式作用因子,如La蛋白、多聚嘧啶串结合蛋白1等的参与下,直接招募核糖体小亚基到mRNA的翻译起始位点,启始翻译.研究发现,参与细胞生长、分化、细胞周期进程、凋亡和压力调控的相关蛋白中通常含有IRES元件.基于功能,我们提出假说:转录激活因子1(ATF1)的5'-UTR可能具有IRES活性.为验证假说,首先构建了含全长ATF1 5'-UTR的双荧光素酶报告质粒;质粒转染结合报告酶活性分析显示,ATF1 5'-UTR在Bel7402、HCT-8和HEK293细胞中表现出不同的IRES活性;而此IRES活性与5'-UTR中的隐藏启动子无关.同时还发现,ATF1 5'-UTR在NIH3T3细胞中却没有IRES活性.与此结果相一致,Western印迹检测ATF1在这几种细胞系中的表达.结果显示,Bel7402、HCT-8和HEK293中ATF1蛋白质表达水平较高,而在NIH3T3中却极低.ATF1 5'-UTR的系列5'-删除突变及报告酶分析证明,ATF1 5'-UTR的完整性对其IRES活性大小发挥重要作用;其中5'端的204 bp序列对其IRES活性贡献较大.RNA-蛋白免疫共沉淀实验揭示,ATF1 5'-UTR可与La和PTBP1蛋白结合;抑制La和PTBP1蛋白质的表达,并可减低HEK293细胞中ATF1蛋白质表达水平.这些结果提示,La和PTBP1蛋白(两种ITAFs)为ATF1 5'-UTR发挥IRES活性所必需.总之,上述结果证明,ATF1 5'-UTR具有IRES活性,其活性发挥依赖与La和PTBP1蛋白的结合.上述发现为进一步研究La和PTBP1表达及亚细胞定位对ATF1 IRES调控机制的影响奠定了基础.The two general 5＇-untranslated region（ 5＇-UTR） translation initiation mechanisms are capdependent and internal ribosome entry site（ IRES） directed mechanisms. IRES trans-acting factors（ ITAFs）,such as the La protein,polypyrimidine tract binding protein 1（ PTBP1）,recruits the 40 S ribosomal subunit to initiates translation at the initiation codon. Many mRNAs coding proteins for cell growth,differentiation,cell cycle progression,apoptosis and stress regulation contain IRES elements.We hypothesized that the 5＇-UTR of activating transcription factor 1（ ATF1） might contain an IRES element. A bicistronic plasmid pRL-ATF1-FL / basic containing the full length 5＇-UTR of ATF1 wasconstructed and transfected into different cell lines. The luciferase activities were detected differently in Bel7402,HCT-8,and HEK293 cells,and did not seem due to the cryptic promoter sequence in the 5＇-UTR. In NIH3T3 cells,no such IRES activity was detected,as shown by the ATF1 protein levels in Western blot. Serial truncation of ATF1 5＇-UTR showed that the integrity of the 5＇-UTR was important for the IRES activity,especially the leading 204 bp. Immunoprecipitation showed that La and PTBP1 bound to the UTR. Inhibition of the La and PTBP1 expression reduced ATF1 expression in HEK293 cells. In conclusion,we showed that ATF1 5＇-UTR exhibited IRES activity and the binding of two ITAFs,La and PTBP1 protein for an IRES activity.

关 键 词：转录激活因子1(ATF1) 5'-非编码区(5'-UTR) 内部核糖体进入位点(IRES) IRES反式作用因子(ITAFs) 

分 类 号：Q522[生物学—生物化学]