Study Of The Internal Ribosome Entry Sites In The 5’UTR Of ATF/CREB Family

ATF/cyclic AMP-responsive element binding protein family play an important role in cell growth, cell proliferation, oncogenesis, immunoregulatory and cellular stress. ATF/C REB family members overexpress in various cancer cells, and affect their growt h and migration. By searching the NCBI database, we found that the 5 ’UTR of ATF/C REB family members are generally long and GC rich, which were accorded with the characteristic of cellular IRES that have been reported. To identify whether the 5’UTR of ATF/C REB family members contain an IRES, a bicistronic plasmid(p RL-FL) which was widely used in detecting IRES activity was constructed. First, the bicistronic plasmid contained the 5’UTR of ATF/C REB family members were contructed to identify their IRES activity. Then, in order to confirm the IRES activity, the preliminary identificated sequence was cloned upstream of the Firefly luciferase reporter and the SV40 promoter was removed. The Renilla and firefly luciferase activities were determined by the Dual Luciferase Reporter Gene Assay K it. The results showed that: ATF1 and ATF2 5’UTR contained IRES and their IRES activity was different in various types of cell lines. To identify the core regions which promote internal initiation of translation, serial truncation of the ATF1 and ATF2 5’UTR was created according to the secondary structure. Results showed that the integrity of ATF1 5’UTR was crucial to its maximized IRES activity, and the 5’ 203 bp secquence were contributed more to its IRES activity. The core region of ATF2 IRES was in the 5’ 30 bp secquence of its 5’UTR.IRES-mediated translation needs participation of IRES trans-acting factors(ITAFs), in order to study the ITAFs which ATF1 and ATF2 IRES needed, this paper confirm four candidate ITAFs(La, PTB, YB1, P97) by consulted some papers. Then RNA-protein immunoprecipitation was performed to study whether ATF1 and/or ATF2 5’UTR interacted with these ITAFs. Results showed that La and PTB bind the ATF1 5’UTR in vivo, while YB1 and P97 did not. These four ITAFs did not bind the ATF2 5’UTR. The study on ATF1 and ATF2 required ITAFs provides a theoretical basis to deep understanding of the difference of IRES activity in different cell types.This paper also treated Bel7402 cells with two chemotherapeutic drugs, paclitaxel and Adriamycin. Study whether the drugs caused cellular stress affect the protein expression and IRES activity of ATF1 and ATF2. The protein expression was detected by Western Blot and IRES activity was determined by the Dual Luciferase Reporter Gene Assay K it. Results showed that ATF1 expression in Bel7402 cells did not affected by drugs caused cellular stress, while during this celluar stress condition, ATF2 protein expression was induced and it was dose and time dependent. ATF2 IRES activity was also induced following treatment with drugs. ATF2 protein expression and IRES activity were induced during cellular stress provides a new insight to study ATF2 function.