Pol III promoters to express small RNAs: Delineation of transcription initiation

Pol III promoters such as U6 are commonly used to express small RNAs, including small interfering RNA, short hairpin RNA, and guide RNA, for the clustered regularly interspaced short palindromic repeats genome-editing system. However, whether the small RNAs were precisely expressed as desired has not been studied. Here, using deep sequencing to analyze small RNAs, we show that, for mouse U6 promoter, sequences immediately upstream of the putative initiation site, which is often modified to accommodate the restriction enzyme sites that enable easy cloning of small RNAs, are critical for precise transcription initiation. When the promoter is kept unmodified, transcription starts precisely from the first available A or G within the range of positions -1 to +2. In addition, we show that transcription from another commonly used pol III promoter, H1, starts at multiple sites, which results in variability at the 5′ end of the transcripts. Thus, inaccuracy of 5′ end of small RNA transcripts might be a common problem when using these promoters to express small RNAs based on currently believed concepts. Our study provides general guidelines for minimizing the variability of initiation, thereby enabling more accurate expression of small RNAs.