RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector.
RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector.
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DOI：
10.1016/j.bbrc.2005.11.037
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发表时间：
2006-01
影响因子：
3.1
通讯作者：
M. Rumi;S. Ishihara;M. Aziz;H. Kazumori;N. Ishimura;T. Yuki;C. Kadota;Y. Kadowaki;Y. Kinoshita
中科院分区：
文献类型：
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作者：
M. Rumi;S. Ishihara;M. Aziz;H. Kazumori;N. Ishimura;T. Yuki;C. Kadota;Y. Kadowaki;Y. Kinoshita
人核糖核酸酶P RNA组分H1、人U6和小鼠U6小核RNA基因的RNA聚合酶III启动子由于其精确的起始和终止位点而常用于短发夹RNA（shRNA）表达载体中。在瞬时转染shRNA载体的过程中，我们观察到H1或U6启动子也表达足够长的转录本，以表达几种报告基因，包括萤火虫荧光素酶、绿色荧光蛋白EGFP和红色荧光蛋白JRed。上游RNA聚合酶II增强子增强了这种较长转录本的表达，下游polyA信号序列完全抑制了这种表达。此外，萤火虫荧光素酶从人类H1启动子的转录是敏感的RNA聚合酶II抑制剂α-鹅膏蕈碱。我们的研究结果表明，shRNA载体中常用的聚合酶III启动子也易于RNA聚合酶II介导的转录，这可能对其靶向使用产生负面影响。
RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use.