In eukaryotes, three RNA polymerases (Pol I, II, and III) are responsible for the transcription of distinct subsets of genes. Gene-external type 3 Pol III promoters use defined transcription start and termination sites, and they are, therefore, widely used for small RNA expression, including short hairpin RNAs in RNAi applications and guide RNAs in CRISPR-Cas systems. We report that all three commonly used human Pol III promoters (7SK, U6, and H1) mediate luciferase reporter gene expression, which indicates Pol II activity, but to a different extent (H1 ≫ U6 > 7SK). We demonstrate that these promoters can recruit Pol II for transcribing extended messenger transcripts. Intriguingly, selective inhibition of Pol II stimulates the Pol III activity and vice versa, suggesting that two polymerase complexes compete for promoter usage. Pol II initiates transcription at the regular Pol III start site on the 7SK and U6 promoters, but Pol II transcription on the most active H1 promoter starts 8 nt upstream of the Pol III start site. This study provides functional evidence for the close relationship of Pol II and Pol III transcription. These mechanistic insights are important for optimal use of Pol III promoters, and they offer additional flexibility for biotechnology applications of these genetic elements.

在真核生物中，三种RNA聚合酶（Pol I，II和III）负责基因的不同子集的转录。基因外部3型Pol III启动子使用定义的转录起始和终止位点，因此，它们广泛用于小RNA表达，包括RNAi应用中的短发夹RNA和CRISPR-Cas系统中的指导RNA。我们报告说，所有三个常用的人类Pol III启动子（7SK，U6和H1）介导萤光素酶报告基因的表达，这表明Pol II的活性，但程度不同（H1→U6> 7SK）。我们证明这些启动子可以招募Pol II转录扩展的信使转录本。有趣的是，对Pol II的选择性抑制会刺激Pol III的活性，反之亦然，这表明两种聚合酶复合物竞争启动子的使用。Pol II在7SK和U6启动子的常规Pol III起始位点启动转录，但在最活跃的H1启动子上的Pol II转录在Pol III起始位点上游8 nt开始。该研究为Pol II和Pol III转录的密切关系提供了功能性证​​据。这些机制的见解对于最佳使用Pol III启动子很重要，并且它们为这些遗传元件的生物技术应用提供了额外的灵活性。