Alu家族是灵长类基因组特有的含量丰富的中度重复序列是短散在.DOC

RNA聚合酶II转录的ALU对PKR 磷酸化的调节作用 杨梅1，沈薇1，吴进峰1，曾贵利1，王峰2，任红3，唐开福3 1 重庆医科大学附属第二医院消化内科 400010 2 四川省华蓥市人民医院 638000 3 重庆医科大学附二院肝炎所 400010 [摘要] 目的：研究ALU自身的RNA聚合酶III启动子对RNA聚合酶II转录的影响，在此基础上探讨RNA聚合酶II转录的ALU是否能够调节PKR的活性。方法：将Alu全基因序列插入pcDNA3.1载体的CMV启动子（一个RNA聚合酶II启动子）的直接下游，构建重组质粒pcDNA3.1-ALU；用重组质粒pcDNA3.1-ALU转染293细胞，提取细胞总RNA，RT-PCR筛选稳定表达ALU的克隆；对稳定表达ALU序列的细胞进行干扰素处理， western-blot法检测PKR的磷酸化水平。结果：Alu全基因序列插入pcDNA3.1载体的CMV启动子直接下游能够被RNA聚合酶II有效转录，说明ALU自身的RNA聚合酶III启动子并不影响RNA聚合酶II的转录。western-blot结果表明：在稳定表达ALU的293细胞中，PKR的磷酸化水平在加干扰素与未加干扰素组均明显高于293细胞对应组。结论：ALU自身的RNA聚合酶III启动子对RNA聚合酶II转录没有明显的影响，但与RNA聚合酶Ⅲ启动下转录的ALU不同，RNA聚合酶II转录的ALU失去了对干扰素的拮抗作用，反而可以通过形成双链RNA的方式激活PKR的活性。 关键词 ALU序列，PKR，RNA聚合酶II，RNA聚合酶III,干扰素 Regulatory role of alu RNA transcripted by RNA polymerase II on PKR phosphorylation YANG Mei1,SHEN Wei1,WU Jing-feng1,ZENG Gui-li1,WANG Feng2,REN Hong3,T-AMG Kai-fu3(1 Department of Gastroenterology,The Second Affiliated Hospital,Chongqing Medical University,400010 2 The Pepole’s Hospital of Huaying ,Shichuang province,638000,3 Institute for Viral Hepatitis,The Second Affiliated Hospital,Chongqing Medical University,400010) Abstract: Objective: To study the influence of the ALUs polymerase III (Pol III) promoter on RNA polymerase II (Pol II) transcription, and the effect of Pol II driven Alu transcript on the activity of protein kinase R (PKR). Methods: Recombinant plasmid pcDNA3.1-ALU was obtained by inserting full length Alu sequence downstream of CMV promoter; Alu-293 cells stably expressing Pol II driven Alu transcript were established. The PKR phosphorylation was detected by western blot. Results: The Alu sequence directly downstream of CMV promoter can be effectly transcripted by RNA polymerase II. Western blot indicated that the phosphoryaltion level of PKR is higher in Alu-293 cells than in 293 cells. Conclusion: Alu’s Pol III promoter has no obvious influence on the RNA polymerase II transcription; Unlike the pol III driven Alu transcript, the Pol II driven Alu transcript can not alle