Regulation of U6 promoter activity by transcriptional interference in viral vector-based RNAi.
The direct negative impact of the transcriptional activity of one component on the second one in cis is referred to as transcriptional interference (TI). U6 is a type III RNA polymerase III promoter commonly used for driving small hairpin RNA (shRNA) expression in vector-based RNAi. In the design and construction of viral vectors, multiple transcription units may be arranged in close proximity in a space-limited vector. Determining if U6 promoter activity can be affected by TI is critical for the expression of target shRNA in gene therapy or loss-of-function studies. In this research, we designed and implemented a modified retroviral system where shRNA and exogenous gene expressions were driven by two independent transcriptional units. We arranged U6 promoter driving shRNA expression and UbiC promoter in two promoter arrangements. In primary macrophages, we found U6 promoter activity was inhibited by UbiC promoter when in the divergent arrangement but not in tandem. In contrast, PKG promoter had no such negative impact. Instead of enhancing U6 promoter activity, CMV enhancer had significant negative impact on U6 promoter activity in the presence of UbiC promoter. Our results indicate that U6 promoter activity can be affected by TI in a proximal promoter-specific and arrangement-dependent manner.
Nie
Linghu
L
Key Laboratory of Genome Sciences and Information, Cancer Biology and Genetics Group, Beijing Institute of Genomics, Chinese Acadamy of Sciences, Beijing 100029, China.
Das Thakur
Meghna
M
Wang
Yumei
Y
Su
Qin
Q
Zhao
Yongliang
Y
Feng
Yunfeng
Y
D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D008957:Models, Genetic; D007652:Oxo-Acid-Lyases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D014158:Transcription, Genetic
D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; D007652:Oxo-Acid-Lyases; C075960:chorismate pyruvate lyase