RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector.
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 alpha-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.
Rumi
Mohammad
M
Second Department of Internal Medicine, Shimane University School of Medicine, 89-1 Enya-Cho, Izumo-shi, Shimane 693-0021, Japan.
Ishihara
Shunji
S
Aziz
Monowar
M
Kazumori
Hideaki
H
Ishimura
Norihisa
N
Yuki
Takafumi
T
Kadota
Chikara
C
Kadowaki
Yasunori
Y
Kinoshita
Yoshikazu
Y
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D051379:Mice; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012320:RNA Polymerase III / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*
D004791:Enzyme Inhibitors; D012313:RNA; D012319:RNA Polymerase II; D012320:RNA Polymerase III