Mutation of nucleotides around the +1 position of type 3 polymerase III promoters: The effect on transcriptional activity and start site usage.
Mutation of nucleotides around the +1 position of type 3 polymerase III promoters: The effect on transcriptional activity and start site usage.
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DOI：
10.1080/21541264.2017.1322170
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Herrera-Carrillo E
中科院分区：
文献类型：
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作者：
Gao Z;Harwig A;Berkhout B;Herrera-Carrillo E
3型RNA聚合酶III（Pol III）启动子广泛用于在流行的RNAi和CRISPR-Cas基因调控系统中表达小RNA，例如短发夹RNA和向导RNA。尽管通常认为3型Pol III启动子使用确定的转录起始位点（+1位置），但大多数人造启动子构建体含有局部序列改变，其对转录效率和起始准确性的影响是未知的。对于三种人3型Pol III启动子（7SK、U6和H1），我们证明了+1位置周围的核苷酸影响转录效率和起始位点选择。在+1位置具有A或G的人7SK和U6启动子有效地产生具有精确的+1起始位点的小RNA。带有+1A或G的人类H1启动子也能有效地产生小RNA，但来自−3/−1窗口中的多个起始位点。这些结果为设计用于研究和治疗目的的精确表达设计的小RNA的载体提供了新的见解。
Type 3 RNA polymerase III (Pol III) promoters are widely used for the expression of small RNAs such as short hairpin RNA and guide RNA in the popular RNAi and CRISPR-Cas gene regulation systems. Although it is generally believed that type 3 Pol III promoters use a defined transcription start site (+1 position), most man-made promoter constructs contain local sequence alterations of which the impact on transcription efficiency and initiation accuracy is not known. For three human type 3 Pol III promoters (7SK, U6, and H1), we demonstrated that the nucleotides around the +1 position affect both the transcriptional efficiency and start site selection. Human 7SK and U6 promoters with A or G at the +1 position efficiently produced small RNAs with a precise +1 start site. The human H1 promoter with +1A or G also efficiently produced small RNAs but from multiple start sites in the −3/−1 window. These results provide new insights for the design of vectors for accurate expression of designed small RNAs for research and therapeutic purposes.