Goguen Ryan P, Del Corpo Olivier, Malard Camille M G, Daher Aïcha, Alpuche-Lazcano Sergio P, Chen Michelle J, Scarborough Robert J, Gatignol Anne
Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada.
Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, Canada.
Mol Ther Nucleic Acids. 2021 Jan 1;23:1020-1034. doi: 10.1016/j.omtn.2020.12.022. eCollection 2021 Mar 5.
The expression of short hairpin RNAs (shRNAs) in cells has many potential therapeutic applications, including as a functional cure for HIV. The RNA polymerase III promoters H1, 7SK, and U6 have all been used to express shRNAs. However, there have been no direct and simultaneous comparisons of shRNA potency, expression level, and transcriptional profile between the promoters. We show that the 7SK and U6 promoters result in higher shRNA levels and potency compared to the H1 promoter but that in transduced T lymphocytes, higher expression levels can also lead to growth defects. We present evidence that Dicer cleavage of shRNAs is measured from the first base pair in the shRNA stem, rather than from the 5' end as previously shown for structurally related microRNAs. As a result, guide-strand identity was unaffected by variations in 5' transcription start sites among the different promoters, making expression levels the main determinant of shRNA potency. While all promoters generated shRNAs with variable start sites, the U6 promoter was the most accurate in using its intended +1 position. Our results have implications for the development of therapeutic small RNAs for gene therapy and for our understanding of how shRNAs are processed in cells.
短发夹RNA（shRNA）在细胞中的表达具有许多潜在的治疗应用，包括作为治疗HIV的功能性疗法。RNA聚合酶III启动子H1、7SK和U6均已用于表达shRNA。然而，尚未对这些启动子之间的shRNA效力、表达水平和转录谱进行直接且同时的比较。我们发现，与H1启动子相比，7SK和U6启动子可产生更高水平的shRNA和更强的效力，但在转导的T淋巴细胞中，较高的表达水平也会导致生长缺陷。我们提供的证据表明，shRNA的Dicer切割是从shRNA茎中的第一个碱基对开始测量的，而不是像之前针对结构相关的微小RNA所显示的那样从5'端开始。因此，引导链的一致性不受不同启动子之间5'转录起始位点变化的影响，使得表达水平成为shRNA效力的主要决定因素。虽然所有启动子都产生了起始位点可变的shRNA，但U6启动子在使用其预期的+1位置时最为准确。我们的结果对用于基因治疗的治疗性小RNA的开发以及我们对shRNA在细胞中如何加工的理解具有启示意义。
